Pharmaceutical formulations comprising high purity cangrelor and methods for preparing and using the same

ABSTRACT

The present invention relates to high purity cangrelor, pharmaceutical formulations comprising high purity cangrelor as an active ingredient, methods for preparing such compounds and formulations, and methods for using the pharmaceutical formulations in the inhibition of platelet activation and aggregation.

CROSS-REFERENCED TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 15/049,727,filed Feb. 22, 2016, which in turn is a continuation of U.S. Ser. No.14/796,368, filed Jul. 10, 2015, now U.S. Pat. No. 9,295,687, whichclaims benefit of U.S. Ser. No. 62/103,136, filed Jan. 14, 2015.

FIELD OF THE INVENTION

The present invention is generally directed towards pharmaceuticalformulations comprising high purity cangrelor or one or more saltsthereof as an active ingredient, to methods for preparing suchpharmaceutical formulations where low levels of impurities areconsistently achieved and maintained, and to methods for using thepharmaceutical formulations in the inhibition of platelet activation andaggregation.

BACKGROUND OF THE INVENTION

The inhibition of platelet activation and aggregation, or antiplatelettherapy, has been recognized as a means to impact coagulation andinflammation in a way that conventional anticoagulant therapy is unableto (Bhatt, D. L.; Topol, E. J. Nat Rev Drug Disc 2003, 2, 15-28). Assuch, inhibitors of platelet activation and aggregation are substancesthat are useful during percutaneous coronary intervention (PCI) andother catherization techniques in order to reduce bleedingcomplications, and in the treatment of acute coronary syndromes (ACS)and clotting disorders in general. One class of antiplatelet agents isinhibitors of the P2Y₁₂ receptor, a G-protein coupled purinergicreceptor which is an important component of platelet activation (Dorsam,R. T.; Kunapuli, S. P. J Clin Invest 2003, 113, 340-345). In particular,cangrelor ([dichloro-[[[(2R,3S,4R,5R)-3,4-dihydroxy-5-[6-(2-methylsulfanylethylamino)-2-(3,3,3-trifluoropropylsulfanyl)purin-9-yl]oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]methyl]phosphonicacid; the mixed mono(anhydride) ofN-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-5′-adenylic acidwith dichloromethylenebisphosphonic acid) is a reversible inhibitor ofthe P2Y₁₂ receptor which is under clinical evaluation for its potentialuse in PCI.

Cangrelor (also referred to as ARC69931MX) is a synthetic analogue ofadenosine triphosphate (ATP) and a potent antagonist of the P2Y₁₂receptor with a pIC₅₀ of 9.35 (Chattaraj, S. C. Curr Opin Investig Drugs2001, 2, 250-55; Diaz-Ricart, M. Drugs Future 2008, 33, 101-110; U.S.Pat. No. 5,721,219 and U.S. Pat. No. 5,955,447). It is being developedas the sodium salt.

In light of the medical and therapeutic applications of cangrelor, it isessential that pharmaceutical formulations comprising cangrelor maintainhigh levels of purity. Formulations comprising cangrelor are compoundedformulations, e.g., cangrelor undergoes a compounding process followingits synthesis so that it is usable and stable for medical andtherapeutic applications. This compounding process typically includesmixing the drug with excipients in a solution, followed by asepticfiltration and lyophilization.

Impurities such as, but not exclusively, dichloromethylenebisphosphonicacid,N-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-5′-adenylic acid(a product of the hydrolysis of the dichloromethylenebisphosphonategroup on cangrelor), its bis(anhydride) withdichloromethylenebisphosphonic acid,N-[2-(methylsulfinyl)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-5′-adenylicacid monoanhydride with dichloromethylenebisphosphonic acid and2-(3,3,3-trifluoropropylthio)-N-(2-(methylthio)ethyl)-adenine and othersmay be generated during the synthesis and the compounding process. Thesecompounds are represented in their neutral form but are generallypresent as salts.

Methods have been developed that minimize the generation of impuritiesduring cangrelor synthesis. However, impurities produced during thecompounding process remain problematic. It has been shown that variouscompounding processes can result in formulations in which a significantproportion of cangrelor has been degraded, which may affect not onlyproduct stability and shelf-life, but ultimately the ability to controldosage during administration to patients. In addition, because thepharmacological impact of the degradation products has not beenevaluated in clinical settings, it is critical to maintain them to alevel at or below the levels used in clinical evaluation. Therefore,development of a compounding process for formulating cangrelor thatconsistently generates formulations having low levels of impurities isdesirable.

The invention disclosed herein addresses the need for pharmaceuticalformulations comprising high purity cangrelor as the active ingredientand methods for producing the same, where low levels of impurities areconsistently achieved and maintained.

SUMMARY OF THE INVENTION

The present invention relates to (i) high purity cangrelor, or one ormore salts thereof, (ii) pharmaceutical formulations comprising highpurity cangrelor, or one or more salts thereof, as an active ingredientand one or more pharmaceutically acceptable excipients, (iii) methodsfor preparing such compounds and formulations, and (iv) methods forusing compounds and the pharmaceutical formulations in the inhibition ofplatelet activation and aggregation.

Thus in one embodiment, the invention relates to high purity cangrelor,or a salt thereof. High purity cangrelor is cangrelor having a combinedtotal of selected hydrolysis and oxidation degradants of cangrelor notexceeding about 1.5% by weight of the high purity cangrelor (i.e., highpurity cangrelor includes (i) cangrelor and (ii) selected hydrolysis andoxidation degradants of cangrelor not exceeding about 1.5% by weight ofthe combination of the cangrelor and the degradants). Selectedhydrolysis and oxidation degradants of cangrelor are impurity A,impurity B, impurity C, impurity D and impurity E. Thus, in one aspectof this embodiment, high purity cangrelor of the present invention has acombined impurity level of impurities A, B, C, D and E of less thanabout 1.5% by weight of the high purity cangrelor. In other aspects,high purity cangrelor of the present invention has a combined impuritylevel of impurities A, B, C, D and E of less than about 1.4% by weight,less than about 1.3% by weight, less than about 1.2% by weight or lessthan about 1.0% by weight. In another aspect, the amount of impurity Apresent in the high purity cangrelor is less than about 0.5% by weight,and/or the amount of impurity B present in the high purity cangrelor isless than about 0.2% by weight, and/or the amount of impurity C presentin the high purity cangrelor is less than about 0.3% by weight, and/orthe amount of impurity D present in the high purity cangrelor is lessthan about 0.2% by weight, and/or the amount of impurity E present inthe high purity cangrelor is less than about 0.5% by weight of the highpurity cangrelor. In one aspect, the amount of impurities A and Dpresent in the high purity cangrelor are each less than about 0.5% byweight of the high purity cangrelor.

In some aspects of this embodiment, the high purity cangrelor is storedin a chemically inert dry gas in a sealed vessel. When present, thechemically inert dry gas is nitrogen or argon.

In some aspects of this embodiment, the high purity cangrelor is storedin a stoppered, sealed dry vessel, wherein components thereof aresufficiently dried to minimize moisture transfer to cangrelor. Inparticular aspects, the stoppered, sealed dry vessel is a lyophilizationvial stoppered with a stopper dried to minimize its own moisture level.

In a second embodiment, the invention relates to a pharmaceuticalformulation comprising high purity cangrelor, or a salt thereof, as anactive ingredient and one or more pharmaceutically acceptableexcipients.

High purity cangrelor is cangrelor having a combined total of selectedhydrolysis and oxidation degradants of cangrelor not exceeding about1.5% by weight of the high purity cangrelor. Selected hydrolysis andoxidation degradants of cangrelor are impurity A, impurity B, impurityC, impurity D and impurity E. Thus, in one aspect of this embodiment,high purity cangrelor of the present invention has a combined impuritylevel of impurities A, B, C, D and E of less than about 1.5% by weightof the high purity cangrelor. In other aspects, high purity cangrelor ofthe present invention has a combined impurity level of impurities A, B,C, D and E of less than about 1.4% by weight, less than about 1.3% byweight, less than about 1.2% by weight or less than about 1.0% byweight. In another aspect, the amount of impurity A present in the highpurity cangrelor is less than about 0.5% by weight, and/or the amount ofimpurity B present in the high purity cangrelor is less than about 0.2%by weight, and/or the amount of impurity C present in the high puritycangrelor is less than about 0.3% by weight, and/or the amount ofimpurity D present in the high purity cangrelor is less than about 0.2%by weight, and/or the amount of impurity E present in the high puritycangrelor is less than about 0.5% by weight of the high puritycangrelor. In one aspect, the amount of impurities A and D present inthe high purity cangrelor are each less than about 0.5% by weight of thehigh purity cangrelor.

In certain aspects of this embodiment, the pharmaceutically acceptableexcipient is a polyol. When present, the polyol is at least one memberselected from the group consisting of mannitol and sorbitol. In oneaspect, the invention relates to a pharmaceutical formulation consistingof high purity cangrelor, or a salt thereof, as an active ingredient andmannitol or sorbitol, or both mannitol and sorbitol.

In certain aspects of this embodiment, the pharmaceutical formulationcomprises about 16-21% of high purity cangrelor, expressed in terms ofthe free acid but present as the free acid or a salt thereof, and about84-79% of the one or more pharmaceutically acceptable excipients, byweight of the pharmaceutical formulation.

In some aspects of this embodiment, the pharmaceutical formulation isstored in a chemically inert dry gas in a sealed vessel. When present,the chemically inert dry gas is nitrogen or argon.

In some aspects of this embodiment, the pharmaceutical formulation isstored in a stoppered, sealed dry vessel, wherein components thereof aresufficiently dried to minimize moisture transfer to a component of thepharmaceutical formulation. In particular aspects, the stoppered, sealeddry vessel is a lyophilization vial stoppered with a stopper dried tominimize its own moisture level.

In a third embodiment, the invention relates to a method for preparinghigh purity cangrelor, or a salt thereof, comprising (a) dissolvingcangrelor or a salt thereof in a solvent to form a first solution; (b)mixing a pH-adjusting agent with the first solution to form a secondsolution, wherein the pH of the second solution is between about 7.0 and9.5; and (c) removing the solvent from the second solution to producehigh purity cangrelor or a salt thereof under conditions wherein a levelof moisture of less than about 2.0% by weight is achieved, therebypreparing high purity cangrelor or a salt thereof. In one aspect, theinvention relates to a method for preparing high purity cangrelor, or asalt thereof, consisting of (a) dissolving cangrelor or a salt thereofin a solvent to form a first solution; (b) mixing a pH-adjusting agentwith the first solution to form a second solution, wherein the pH of thesecond solution is between about 7.0 and 9.5; and (c) removing thesolvent from the second solution to produce high purity cangrelor or asalt thereof under conditions wherein a level of moisture of less thanabout 2.0% by weight is achieved, thereby preparing high puritycangrelor or a salt thereof.

High purity cangrelor is cangrelor having a combined total of selectedhydrolysis and oxidation degradants of cangrelor not exceeding about1.5% by weight of the high purity cangrelor. Selected hydrolysis andoxidation degradants of cangrelor are impurity A, impurity B, impurityC, impurity D and impurity E. Thus, in one aspect of this embodiment,high purity cangrelor of the present invention has a combined impuritylevel of impurities A, B, C, D and E of less than about 1.5% by weightof the high purity cangrelor. In other aspects, high purity cangrelor ofthe present invention has a combined impurity level of impurities A, B,C, D and E of less than about 1.4% by weight, less than about 1.3% byweight, less than about 1.2% by weight or less than about 1.0% byweight. In another aspect, the amount of impurity A present in the highpurity cangrelor is less than about 0.5% by weight, and/or the amount ofimpurity B present in the high purity cangrelor is less than about 0.2%by weight, and/or the amount of impurity C present in the high puritycangrelor is less than about 0.3% by weight, and/or the amount ofimpurity D present in the high purity cangrelor is less than about 0.2%by weight, and/or the amount of impurity E present in the high puritycangrelor is less than about 0.5% by weight of the high puritycangrelor. In one aspect, the amount of impurities A and D present inthe high purity cangrelor are each less than about 0.5% by weight of thehigh purity cangrelor.

In some aspects of this embodiment, the pH of the second solution isabout 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5,between about 7.0 and 8.0, between about 7.5 and 8.5, between about 8.0and 9.0, or between about 8.5 and 9.5.

In some aspects of this embodiment, mixing of (b) is achieved by addingthe pH-adjusting agent to the first solution. In other aspects of thisembodiment, the mixing of (b) is achieved by adding the first solutionto the pH-adjusting agent. In further aspects of this embodiment, themixing of (b) is achieved by simultaneous combination of thepH-adjusting agent and the first solution. In some of these aspects, thepH-adjusting agent is added to the first solution in portions. In otheraspects, the pH-adjusting agent is added to the first solution at aconstant rate. In some aspects of this embodiment, mixing is achieved byusing one or more mixing devices. When used, the mixing device isselected from the group consisting of a paddle mixer, magnetic stirrer,shaker, re-circulating pump, homogenizer, and any combination thereof.Alternatively, the mixing device is a homogenizer, a bottom mountmagnetic device, a paddle mixer, or a combination thereof. In furtheraspects of this embodiment, the mixing is achieved through high shearmixing.

In some aspects of this embodiment, removing the solvent (c) is throughlyophilization.

In some aspects of this embodiment, one or more of the steps isperformed in the absence of light, such as the mixing of (b).

In some aspects of this embodiment, one or more of the steps isperformed under a chemically inert gas, in particular nitrogen, such asthe mixing of (b).

In some aspects of this embodiment, the method further comprisessterilizing the second solution after the mixing of (b) and before theremoval of the solvent. In one aspect, sterilization is achieved byaseptic filtration.

In some aspects of this embodiment, the method further comprises storingthe high purity cangrelor or salt thereof in a chemically inert dry gasin a sealed vessel. When present, the chemically inert dry gas isnitrogen or argon.

In some aspects of this embodiment, the method further comprises storingthe high purity cangrelor or salt thereof in a stoppered, sealed dryvessel, wherein components thereof are sufficiently dried to minimizemoisture transfer to cangrelor. In particular aspects, the stoppered,sealed dry vessel is a lyophilization vial stoppered with a stopperdried to minimize its own moisture level.

In a fourth embodiment, the invention relates to a method for preparinga pharmaceutical formulation comprising high purity cangrelor, or a saltthereof, as an active ingredient and one or more pharmaceuticallyacceptable excipients, comprising (a) dissolving cangrelor or a saltthereof in a solvent to form a first solution; (b) mixing a pH-adjustingagent with the first solution to form a second solution, wherein the pHof the second solution is between about 7.0 and 9.5; and (c) removingthe solvent from the second solution to produce high purity cangrelor ora salt thereof under conditions wherein a level of moisture of less thanabout 2.0% by weight is achieved, wherein one or more pharmaceuticallyacceptable excipients is added to the first solution, or to the secondsolution, or to both, thereby preparing a pharmaceutical formulationcomprising high purity cangrelor or a salt thereof.

High purity cangrelor is cangrelor having a combined total of selectedhydrolysis and oxidation degradants of cangrelor not exceeding about1.5% by weight of the high purity cangrelor. Selected hydrolysis andoxidation degradants of cangrelor are impurity A, impurity B, impurityC, impurity D and impurity E. Thus, in one aspect of this embodiment,high purity cangrelor of the present invention has a combined impuritylevel of impurities A, B, C, D and E of less than about 1.5% by weightof the high purity cangrelor. In other aspects, high purity cangrelor ofthe present invention has a combined impurity level of impurities A, B,C, D and E of less than about 1.4% by weight, less than about 1.3% byweight, less than about 1.2% by weight or less than about 1.0% byweight. In another aspect, the amount of impurity A present in the highpurity cangrelor is less than about 0.5% by weight, and/or the amount ofimpurity B present in the high purity cangrelor is less than about 0.2%by weight, and/or the amount of impurity C present in the high puritycangrelor is less than about 0.3% by weight, and/or the amount ofimpurity D present in the high purity cangrelor is less than about 0.2%by weight, and/or the amount of impurity E present in the high puritycangrelor is less than about 0.5% by weight of the high puritycangrelor. In one aspect, the amount of impurities A and D present inthe high purity cangrelor are each less than about 0.5% by weight of thehigh purity cangrelor.

In some aspects of this embodiment, the pH of the second solution isabout 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5,between about 7.0 and 8.0, between about 7.5 and 8.5, between about 8.0and 9.0, or between about 8.5 and 9.5.

In some aspects of this embodiment, mixing of (b) is achieved by addingthe pH-adjusting agent to the first solution. In other aspects of thisembodiment, the mixing of (b) is achieved by adding the first solutionto the pH-adjusting agent. In further aspects of this embodiment, themixing of (b) is achieved by simultaneous combination of thepH-adjusting agent and the first solution. In some of these aspects, thepH-adjusting agent is added to the first solution in portions. In otheraspects, the pH-adjusting agent is added to the first solution at aconstant rate. In some aspects of this embodiment, mixing is achieved byusing one or more mixing devices. When used, the mixing device isselected from the group consisting of a paddle mixer, magnetic stirrer,shaker, re-circulating pump, homogenizer, and any combination thereof.Alternatively, the mixing device is a homogenizer, a bottom mountmagnetic device, a paddle mixer, or a combination thereof. In furtheraspects of this embodiment, the mixing is achieved through high shearmixing.

In certain aspects of this embodiment, the pharmaceutically acceptableexcipient is a polyol. When present, the polyol is at least one memberselected from the group consisting of mannitol and sorbitol. In oneaspect, the one or more pharmaceutically acceptable excipient ismannitol or sorbitol, or both mannitol and sorbitol, and the excipientis added to the first solution. In another aspect, the one or morepharmaceutically acceptable excipient is mannitol or sorbitol, or bothmannitol and sorbitol, and the excipient is added to the secondsolution. In one aspect, the invention relates to a method for preparinga pharmaceutical formulation consisting of high purity cangrelor, or asalt thereof, as an active ingredient and mannitol or sorbitol, or bothmannitol and sorbitol, as a pharmaceutically acceptable excipient,comprising (a) dissolving cangrelor or a salt thereof in a solvent toform a first solution; (b) mixing a pH-adjusting agent with the firstsolution to form a second solution, wherein the pH of the secondsolution is between about 7.0 and 9.5; and (c) removing the solvent fromthe second solution to produce high purity cangrelor or a salt thereofunder conditions wherein a level of moisture of less than about 2.0% byweight is achieved, wherein the pharmaceutically acceptable excipient isadded to the first solution, or to the second solution, or to both,thereby preparing a pharmaceutical formulation comprising high puritycangrelor or a salt thereof. In another aspect, the invention relates toa method for preparing a pharmaceutical formulation consisting of highpurity cangrelor, or a salt thereof, as an active ingredient andmannitol or sorbitol, or both mannitol and sorbitol, as apharmaceutically acceptable excipient, consisting of (a) dissolvingcangrelor or a salt thereof in a solvent to form a first solution; (b)mixing a pH-adjusting agent with the first solution to form a secondsolution, wherein the pH of the second solution is between about 7.0 and9.5; and (c) removing the solvent from the second solution to producehigh purity cangrelor or a salt thereof under conditions wherein a levelof moisture of less than about 2.0% by weight is achieved, wherein thepharmaceutically acceptable excipient is added to the first solution, orto the second solution, or to both, thereby preparing a pharmaceuticalformulation comprising high purity cangrelor or a salt thereof.

In certain aspects of this embodiment, the pharmaceutical formulationcomprises about 16-21% of high purity cangrelor, expressed as the freeacid but present as the free acid or a salt thereof, and about 84-79% ofthe one or more pharmaceutically acceptable excipients, by weight of thepharmaceutical formulation.

In some aspects of this embodiment, removing the solvent (c) is throughlyophilization.

In some aspects of this embodiment, one or more of the steps isperformed in the absence of light, such as the mixing of (b).

In some aspects of this embodiment, one or more of the steps isperformed under a chemically inert gas, including nitrogen, such as themixing of (b).

In some aspects of this embodiment, the method further comprisessterilizing the second solution after the mixing of (b) and before theremoval of the solvent. In one aspect, sterilization is achieved byaseptic filtration.

In some aspects of this embodiment, the method further comprises storingthe formulation in a chemically inert dry gas in a sealed vessel. Whenpresent, the chemically inert dry gas is nitrogen or argon.

In some aspects of this embodiment, the method further comprises storingthe formulation in a stoppered, sealed dry vessel, wherein componentsthereof are sufficiently dried to minimize moisture transfer to acomponent of the pharmaceutical formulation. In particular aspects, thestoppered, sealed dry vessel is a lyophilization vial stoppered with astopper dried to minimize its own moisture level.

In a fifth embodiment, the invention relates to high purity cangrelor,or a salt thereof, prepared by a method comprising (a) dissolvingcangrelor or a salt thereof in a solvent to form a first solution; (b)mixing a pH-adjusting agent with the first solution to form a secondsolution, wherein the pH of the second solution is between about 7.0 and9.5; and (c) removing the solvent from the second solution to producehigh purity cangrelor or a salt thereof under conditions wherein a levelof moisture of less than about 2.0% by weight is achieved. In oneaspect, the invention relates to high purity cangrelor, or a saltthereof, prepared by a method consisting of (a) dissolving cangrelor ora salt thereof in a solvent to form a first solution; (b) mixing apH-adjusting agent with the first solution to form a second solution,wherein the pH of the second solution is between about 7.0 and 9.5; and(c) removing the solvent from the second solution to produce high puritycangrelor or a salt thereof under conditions wherein a level of moistureof less than about 2.0% by weight is achieved.

High purity cangrelor is cangrelor having a combined total of selectedhydrolysis and oxidation degradants of cangrelor not exceeding about1.5% by weight of the high purity cangrelor. Selected hydrolysis andoxidation degradants of cangrelor are impurity A, impurity B, impurityC, impurity D and impurity E. Thus, in one aspect of this embodiment,high purity cangrelor of the present invention has a combined impuritylevel of impurities A, B, C, D and E of less than about 1.5% by weightof the high purity cangrelor. In other aspects, high purity cangrelor ofthe present invention has a combined impurity level of impurities A, B,C, D and E of less than about 1.4% by weight, less than about 1.3% byweight, less than about 1.2% by weight or less than about 1.0% byweight. In another aspect, the amount of impurity A present in the highpurity cangrelor is less than about 0.5% by weight, and/or the amount ofimpurity B present in the high purity cangrelor is less than about 0.2%by weight, and/or the amount of impurity C present in the high puritycangrelor is less than about 0.3% by weight, and/or the amount ofimpurity D present in the high purity cangrelor is less than about 0.2%by weight, and/or the amount of impurity E present in the high puritycangrelor is less than about 0.5% by weight of the high puritycangrelor. In one aspect, the amount of impurities A and D present inthe high purity cangrelor are each less than about 0.5% by weight of thehigh purity cangrelor.

In some aspects of this embodiment, the pH of the second solution isabout 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5,between about 7.0 and 8.0, between about 7.5 and 8.5, between about 8.0and 9.0, or between about 8.5 and 9.5.

In some aspects of this embodiment, mixing of (b) is achieved by addingthe pH-adjusting agent to the first solution. In other aspects of thisembodiment, the mixing of (b) is achieved by adding the first solutionto the pH-adjusting agent. In further aspects of this embodiment, themixing of (b) is achieved by simultaneous combination of thepH-adjusting agent and the first solution. In some of these aspects, thepH-adjusting agent is added to the first solution in portions. In otheraspects, the pH-adjusting agent is added to the first solution at aconstant rate. In some aspects of this embodiment, mixing is achieved byusing one or more mixing devices. When used, the mixing device isselected from the group consisting of a paddle mixer, magnetic stirrer,shaker, re-circulating pump, homogenizer, and any combination thereof.Alternatively, the mixing device is a homogenizer, a bottom mountmagnetic device, a paddle mixer, or a combination thereof. In furtheraspects of this embodiment, the mixing is achieved through high shearmixing.

In some aspects of this embodiment, removing the solvent (c) is throughlyophilization.

In some aspects of this embodiment, one or more of the steps isperformed in the absence of light, such as the mixing of (b).

In some aspects of this embodiment, one or more of the steps isperformed under nitrogen, such as the mixing of (b).

In some aspects of this embodiment, the method further comprisessterilizing the second solution after the mixing of (b) and before theremoval of the solvent. In one aspect, sterilization is achieved byaseptic filtration.

In some aspects of this embodiment, the method further comprises storingthe high purity cangrelor or salt thereof in a chemically inert dry gasin a sealed vessel. When present, the chemically inert dry gas isnitrogen or argon.

In some aspects of this embodiment, the method further comprises storingthe high purity cangrelor or salt thereof in a stoppered, sealed dryvessel, wherein components thereof are sufficiently dried to minimizemoisture transfer to cangrelor. In particular aspects, the stoppered,sealed dry vessel is a lyophilization vial stoppered with a stopperdried to minimize its own moisture level.

In a sixth embodiment, the invention relates to a pharmaceuticalformulation comprising high purity cangrelor, or a salt thereof, as anactive ingredient and one or more pharmaceutically acceptable excipientsprepared by a method comprising (a) dissolving cangrelor or a saltthereof in a solvent to form a first solution; (b) mixing a pH-adjustingagent with the first solution to form a second solution, wherein the pHof the second solution is between about 7.0 and 9.5; and (c) removingthe solvent from the second solution to produce high purity cangrelor ora salt thereof under conditions wherein a level of moisture of less thanabout 2.0% by weight is achieved, wherein one or more pharmaceuticallyacceptable excipients is added to the first solution, or to the secondsolution, or to both.

High purity cangrelor is cangrelor having a combined total of selectedhydrolysis and oxidation degradants of cangrelor not exceeding about1.5% by weight of the high purity cangrelor. Selected hydrolysis andoxidation degradants of cangrelor are impurity A, impurity B, impurityC, impurity D and impurity E. Thus, in one aspect of this embodiment,high purity cangrelor of the present invention has a combined impuritylevel of impurities A, B, C, D and E of less than about 1.5% by weightof the high purity cangrelor. In other aspects, high purity cangrelor ofthe present invention has a combined impurity level of impurities A, B,C, D and E of less than about 1.4% by weight, less than about 1.3% byweight, less than about 1.2% by weight or less than about 1.0% byweight. In another aspect, the amount of impurity A present in the highpurity cangrelor is less than about 0.5% by weight, and/or the amount ofimpurity B present in the high purity cangrelor is less than about 0.2%by weight, and/or the amount of impurity C present in the high puritycangrelor is less than about 0.3% by weight, and/or the amount ofimpurity D present in the high purity cangrelor is less than about 0.2%by weight, and/or the amount of impurity E present in the high puritycangrelor is less than about 0.5% by weight of the high puritycangrelor. In one aspect, the amount of impurities A and D present inthe high purity cangrelor are each less than about 0.5% by weight of thehigh purity cangrelor.

In some aspects of this embodiment, the pH of the second solution isabout 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5,between about 7.0 and 8.0, between about 7.5 and 8.5, between about 8.0and 9.0, or between about 8.5 and 9.5.

In some aspects of this embodiment, mixing of (b) is achieved by addingthe pH-adjusting agent to the first solution. In other aspects of thisembodiment, the mixing of (b) is achieved by adding the first solutionto the pH-adjusting agent. In further aspects of this embodiment, themixing of (b) is achieved by simultaneous combination of thepH-adjusting agent and the first solution. In some of these aspects, thepH-adjusting agent is added to the first solution in portions. In otheraspects, the pH-adjusting agent is added to the first solution at aconstant rate. In some aspects of this embodiment, mixing is achieved byusing one or more mixing devices. When used, the mixing device isselected from the group consisting of a paddle mixer, magnetic stirrer,shaker, re-circulating pump, homogenizer, and any combination thereof.Alternatively, the mixing device is a homogenizer, a bottom mountmagnetic device, a paddle mixer, or a combination thereof. In furtheraspects of this embodiment, the mixing is achieved through high shearmixing.

In certain aspects of this embodiment, the pharmaceutically acceptableexcipient is a polyol. When present, the polyol is at least one memberselected from the group consisting of mannitol and sorbitol. In oneaspect, the one or more pharmaceutically acceptable excipient ismannitol or sorbitol, or both mannitol and sorbitol, and the excipientis added to the first solution. In another aspect, the one or morepharmaceutically acceptable excipient is mannitol or sorbitol, or bothmannitol and sorbitol, and the excipient is added to the secondsolution. In one aspect, the invention relates to a pharmaceuticalformulation consisting of high purity cangrelor, or a salt thereof, asan active ingredient and mannitol or sorbitol, or both mannitol andsorbitol, as a pharmaceutically acceptable excipient, prepared by amethod comprising (a) dissolving cangrelor or a salt thereof in asolvent to form a first solution; (b) mixing a pH-adjusting agent withthe first solution to form a second solution, wherein the pH of thesecond solution is between about 7.0 and 9.5; and (c) removing thesolvent from the second solution to produce high purity cangrelor or asalt thereof under conditions wherein a level of moisture of less thanabout 2.0% by weight is achieved, wherein the pharmaceuticallyacceptable excipient is added to the first solution, or to the secondsolution, or to both. In another aspect, the invention relates to apharmaceutical formulation consisting of high purity cangrelor, or asalt thereof, as an active ingredient and mannitol or sorbitol, or bothmannitol and sorbitol, prepared by a method consisting of (a) dissolvingcangrelor or a salt thereof in a solvent to form a first solution; (b)mixing a pH-adjusting agent with the first solution to form a secondsolution, wherein the pH of the second solution is between about 7.0 and9.5; and (c) removing the solvent from the second solution to producehigh purity cangrelor or a salt thereof under conditions wherein a levelof moisture of less than about 2.0% by weight is achieved, wherein thepharmaceutically acceptable excipient is added to the first solution, orto the second solution, or to both.

In certain aspects of this embodiment, the pharmaceutical formulationcomprises about 16-21% of high purity cangrelor, expressed as the freeacid but present as the free acid or a salt thereof, and about 84-79% ofthe one or more pharmaceutically acceptable excipients, by weight of thepharmaceutical formulation.

In some aspects of this embodiment, removing the solvent (c) is throughlyophilization.

In some aspects of this embodiment, one or more of the steps isperformed in the absence of light, such as the mixing of (b).

In some aspects of this embodiment, one or more of the steps isperformed under nitrogen, such as the mixing of (b).

In some aspects of this embodiment, the method further comprisessterilizing the second solution after the mixing of (b) and before theremoval of the solvent. In one aspect, sterilization is achieved byaseptic filtration.

In some aspects of this embodiment, the method further comprises storingthe formulation in a chemically inert dry gas in a sealed vessel. Whenpresent, the chemically inert dry gas is nitrogen or argon.

In some aspects of this embodiment, the method further comprises storingthe formulation in a stoppered, sealed dry vessel, wherein componentsthereof are sufficiently dried to minimize moisture transfer to acomponent of the pharmaceutical formulation. In particular aspects, thestoppered, sealed dry vessel is a lyophilization vial stoppered with astopper dried to minimize its own moisture level.

In a seventh embodiment, the invention relates to a method of inhibitingplatelet activation, aggregation, or both, comprising contactingplatelets with an effective amount of a high purity cangrelor, or a saltthereof, thereby inhibiting platelet activation, aggregation, or both.The method is practiced in vitro, in vivo or ex vivo.

In an eighth embodiment, the invention relates to a method of inhibitingplatelet granule release, comprising contacting platelets with aneffective amount of a high purity cangrelor, or a salt thereof, therebyinhibiting platelet granule release. The method is practiced in vitro,in vivo or ex vivo.

In a ninth embodiment, the invention relates to a method of inhibitingplatelet-leukocyte aggregation, comprising contacting platelets with aneffective amount of a high purity cangrelor, or a salt thereof, therebyinhibiting platelet-leukocyte aggregation. The method is practiced invitro, in vivo or ex vivo.

In a tenth embodiment, the invention relates to a method of inhibitingplatelet-granulocyte aggregation, comprising contacting platelets withan effective amount of a high purity cangrelor, or a salt thereof,thereby inhibiting platelet-granulocyte aggregation. The method ispracticed in vitro, in vivo or ex vivo.

In a eleventh embodiment, the invention relates to a method ofinhibiting platelet loss from the blood, comprising contacting plateletswith an effective amount of a high purity cangrelor, or a salt thereof,thereby inhibiting platelet loss from the blood. The method is practicedin vitro, in vivo or ex vivo.

In a twelfth embodiment, the invention relates to a method of inhibitingplatelet activation, aggregation, or both, in a subject, comprisingadministering an effective amount of a pharmaceutical formulation of thepresent invention to a subject in need thereof, thereby inhibitingplatelet activation, aggregation, or both, in a subject. In certainaspects, the subject may be undergoing percutaneous coronaryintervention (PCI) or a catherization technique, or treatment for acutecoronary syndromes (ACS) or a clotting disorder in general. In otheraspects, the subject is undergoing an ECC-based medical procedure, ahypothermia-based medical procedure, or a hypothermic ECC-based medicalprocedure.

In a thirteenth embodiment, the invention relates to a method ofinhibiting platelet granule release in a subject, comprisingadministering an effective amount of a pharmaceutical formulation of thepresent invention to a subject in need thereof, thereby inhibitingplatelet granule release in a subject. In certain aspects, the subjectmay be undergoing percutaneous coronary intervention (PCI) or acatherization technique, or treatment for acute coronary syndromes (ACS)or a clotting disorder in general. In other aspects, the subject isundergoing an ECC-based medical procedure, a hypothermia-based medicalprocedure, or a hypothermic ECC-based medical procedure.

In a fourteenth embodiment, the invention relates to a method ofinhibiting platelet-leukocyte aggregation in a subject, comprisingadministering an effective amount of a pharmaceutical formulation of thepresent invention to a subject in need thereof, thereby inhibitingplatelet-leukocyte aggregation in a subject. In certain aspects, thesubject may be undergoing percutaneous coronary intervention (PCI) or acatherization technique, or treatment for acute coronary syndromes (ACS)or a clotting disorder in general. In other aspects, the subject isundergoing an ECC-based medical procedure, a hypothermia-based medicalprocedure, or a hypothermic ECC-based medical procedure.

In a fifteenth embodiment, the invention relates to a method ofinhibiting platelet-granulocyte aggregation in a subject, comprisingadministering an effective amount of a pharmaceutical formulation of thepresent invention to a subject in need thereof, thereby inhibitingplatelet-granulocyte aggregation in a subject. In certain aspects, thesubject may be undergoing percutaneous coronary intervention (PCI) or acatherization technique, or treatment for acute coronary syndromes (ACS)or a clotting disorder in general. In other aspects, the subject isundergoing an ECC-based medical procedure, a hypothermia-based medicalprocedure, or a hypothermic ECC-based medical procedure.

In a sixteenth embodiment, the invention relates to a method ofinhibiting platelet loss from the blood of a subject, comprisingadministering an effective amount of a pharmaceutical formulation of thepresent invention to a subject in need thereof, thereby inhibitingplatelet loss from the blood of a subject. In certain aspects, thesubject may be undergoing percutaneous coronary intervention (PCI) or acatherization technique, or treatment for acute coronary syndromes (ACS)or a clotting disorder in general. In other aspects, the subject isundergoing an ECC-based medical procedure, a hypothermia-based medicalprocedure, or a hypothermic ECC-based medical procedure.

In a seventeenth embodiment, the invention relates to a method oftreating or preventing stent thrombosis in a subject, comprisingadministering an effective amount of a pharmaceutical formulation of thepresent invention to a subject in need thereof, thereby treating orpreventing stent thrombosis in a subject. In some aspects of theembodiment, a second anti-thrombotic agent is administered with thepharmaceutical formulation, sequentially or concurrently. In aparticular aspect, the second anti-thrombotic agent is bivalirudin.

In an eighteenth embodiment, the invention relates to a method ofreducing mortality in a subject undergoing stent implantation,comprising administering an effective amount of a pharmaceuticalformulation of the present invention to a subject in need thereof,thereby reducing mortality in a subject undergoing stent implantation.In some aspects of the embodiment, a second anti-thrombotic agent isadministered with the pharmaceutical formulation, sequentially orconcurrently. In a particular aspect, the second anti-thrombotic agentis bivalirudin.

In a nineteenth embodiment, the invention relates to method of treatingor preventing myocardial infarction in a subject, comprisingadministering an effective amount of a pharmaceutical formulation of thepresent invention to a subject in need thereof, thereby treating orpreventing myocardial infarction in a subject. In some aspects of theembodiment, a second anti-thrombotic agent is administered with thepharmaceutical formulation, sequentially or concurrently. In aparticular aspect, the second anti-thrombotic agent is bivalirudin.

In a twentieth embodiment, the invention relates to method of reducingmortality in a subject experiencing myocardial infarction, comprisingadministering an effective amount of a pharmaceutical formulation of thepresent invention to a subject in need thereof, thereby reducingmortality in a subject experiencing myocardial infarction. In someaspects of the embodiment, a second anti-thrombotic agent isadministered with the pharmaceutical formulation, sequentially orconcurrently. In a particular aspect, the second anti-thrombotic agentis bivalirudin.

In a twenty-first embodiment, the invention relates to a medicamentcomprising an effective amount of high purity cangrelor, or a saltthereof, and one or more pharmaceutically acceptable excipients usefulfor treating or preventing stent thrombosis, treating or preventingmyocardial infarction, reducing mortality in a subject undergoing stentimplantation, or reducing mortality in a subject experiencing myocardialinfarction.

DETAILED DESCRIPTION

The present invention relates to (i) high purity cangrelor, or one ormore salts thereof, (ii) pharmaceutical formulations comprising highpurity cangrelor, or one or more salts thereof, as an active ingredientand one or more pharmaceutically acceptable excipients, (iii) methodsfor preparing such compounds and formulations, and (iv) methods forusing high purity cangrelor and the pharmaceutical formulations in theinhibition of platelet activation and aggregation and methods of medicaltreatment of subjects.

Cangrelor (Formula I, also referred to as ARC69931MX) has the IUPACchemical name[dichloro-[[[(2R,3S,4R,5R)-3,4-dihydroxy-5-[6-(2-methylsulfanylethylamino)-2-(3,3,3-trifluoropropylsulfanyl)purin-9-yl]oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]methyl]phosphonicacid and may also be referred to the mixed mono(anhydride) ofN-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-5′-adenylic acidwith dichloromethylenebisphosphonic acid. It is represented in itsneutral form, but it is generally used in a pharmaceutical formulationas a salt, such as the tetrasodium salt. Other salts that may be used inpharmaceutical formulations include other alkali metal salts, e.g.lithium and potassium salts; ammonium salts; alkaline earth metal salts,e.g. calcium and magnesium salts and salts of the Group III elements,e.g. aluminum salts. Salts with suitable organic bases, for example,salts with hydroxylamine; lower alkylamines, e.g. methylamine orethylamine; with substituted lower alkylamines, e.g. hydroxy substitutedalkylamines; or with monocyclic nitrogen heterocyclic compounds, e.g.piperidine or morpholine; and salts with amino acids, e.g. witharginine, lysine etc, or an N-alkyl derivative thereof; or with anaminosugar, e.g. N-methyl-D-glucamine or glucosamine. Non-toxic,physiologically acceptable salts are preferred.

As used herein, reference to cangrelor should be understood to includeboth cangrelor in a neutral form, as well as one or more salts ofcangrelor. Similarly, reference herein to high purity cangrelor shouldbe understood to include both high purity cangrelor in a neutral form,as well as one or more salts of high purity cangrelor.

Methods for the synthesis of cangrelor are known in the art anddescribed in, for example, U.S. Pat. No. 5,721,219 and U.S. Pat. No.5,955,447, both of which are incorporated herein by reference in theirentirety.

Cangrelor is a synthetic analogue of adenosine triphosphate (ATP) and apotent antagonist of the P2Y₁₂ receptor, a G-protein coupled purinergicreceptor which is an important component of platelet activation (Dorsam,R. T.; Kunapuli, S. P. J Clin Invest 2003, 113, 340-345), with a pIC₅₀of 9.35 (Chattaraj, S. C. Curr Opin Investig Drugs 2001, 2, 250-55;Diaz-Ricart, M. Drugs Future 2008, 33, 101-110). Inhibitors of plateletactivation and aggregation are substances that are useful duringpercutaneous coronary intervention (PCI) and other catherizationtechniques in order to reduce bleeding complications, and in thetreatment of acute coronary syndromes (ACS) and clotting disorders ingeneral. The inhibition of platelet activation and aggregation, orantiplatelet therapy, has been recognized as a means to impactcoagulation and inflammation in a way that conventional anticoagulanttherapy is unable to (Bhatt, D. L.; Topol, E. J. Nat Rev Drug Disc 2003,2, 15-28).

Cangrelor can be degraded to a number of impurities, including thefollowing five impurities. Cangrelor can be degraded todichloromethylenebisphosphonic acid (impurity E, Formula VI) andN-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-5′-adenylic acid(impurity A, Formula II) through the hydrolysis of the methylphosphonylphosphate group (a mixed anhydride) or to(3,3,3-trifluoropropylthio)-N-(2-(methylthio)ethyl)-adenine (impurity D,Formula V) through the hydrolysis of the ribofuranoside. The firstprocess is expected to be base catalyzed as is the hydrolysis of ananhydride and the second process is expected to be acid catalyzed as isthe hydrolysis of a glycoside. Other degradants are also postulated tobe generated through hydrolysis, such asN-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-5′-adenylic acidbis(anhydride) bonded to dichloromethylenebisphosphonic acid (impurityB, Formula III) which may form via the hydrolysis of cangrelor toimpurity A followed by addition reaction with a second molecule ofcangrelor. Other degradants result from non-hydrolytic processes, suchasN-[2-(methylsulfinyl)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-5′-adenylicacid monoanhydride bonded to dichloromethylenebisphosphonic acid(impurity C, Formula IV) which clearly occurs by oxidation of cangrelor.These degradants are found as impurities in cangrelor. Other impuritiesmay be generated during the synthesis and the processing of cangrelor aswell.

Those skilled in the art will also immediately recognize that impuritiesA, B, D and E are products of the hydrolysis of cangrelor whereasimpurity C is the product of an oxidation of cangrelor. They will alsorecognize that the nature of cangrelor as an anhydride will result insome measure of reactivity towards water. These impurities will begenerated from high purity cangrelor on handling and storage over timedue to the presence of oxygen and water, present either as a solvent oras moisture. It is therefore critical that processes be put in place tomanufacture pharmaceutical compositions of cangrelor with sufficientlyhigh purity to be generated, stored and administered to patients.

The term “drug product” herein refers to an active ingredient of apharmaceutical formulation. Thus, as used herein a drug product includescangrelor, high purity cangrelor and all of the salts thereof.

Compounding Process for Preparing High Purity Cangrelor andPharmaceutical Formulations Thereof

High purity cangrelor, and salts thereof, and pharmaceuticalformulations comprising the same are produced using a novel compoundingprocess.

1) Dissolving Cangrelor in a Solvent to Form a Cangrelor Solution

In the compounding process of the present invention, cangrelor isdissolved in a solvent or a solvent mixture to form a cangrelorsolution. Cangrelor may be commercially purchased or synthesized byvarious procedures as exemplified in U.S. Pat. No. 5,721,219 and U.S.Pat. No. 5,955,447. The concentration of cangrelor in the solvent mayvary but it will generally be between about 0.5 mg/mL and about 100mg/mL, preferably between about 1 mg/mL and about 50 mg/mL. Inparticular aspects, the concentration of cangrelor in the solvent isabout 1 mg/mL, about 5 mg/mL, about 10 mg/mL, about 20 mg/mL, about 30mg/mL, about 40 mg/mL, about 50 mg/mL, or about 60 mg/mL.

Solvents include aqueous and non-aqueous liquids, including but notlimited to, mono- and di-alcohols such as methanol, ethanol, isopropylalcohol, and propylene glycol; polyhydric alcohols such as glycerol andpolyethylene glycol; buffers; and water. In a specific aspect, a 30mg/mL solution of cangrelor in methanol is prepared. In another specificaspect, a 17 mg/mL solution of cangrelor in water is prepared.

Cangrelor can be dissolved in the solvent by methods known in the art,such as by adding cangrelor to the solvent. For example, cangrelor maybe added to the solvent rapidly, slowly, in portions, at a constantrate, at a variable rate, or a combination thereof. A mixing deviceknown in the art may be used to dissolve cangrelor. Examples of mixingdevices include, but are not limited to, a paddle mixer, magneticstirrer, shaker, re-circulating pump, bottom mount magnetic mixer,homogenizer, and any combination thereof. Suitable mixing rates willdepend on such factors as the identity of the solvent, the desired finalconcentration, and the identity of the mixing device. However, suitablemixing rates include between about 50 and about 2000 rpm, such asbetween about 300 and about 1500 rpm. Dissolution may be performed atroom temperature, at elevated temperature or at decreased temperatureusing techniques to control temperature known in the art. Preferably,the dissolution is performed at or below room temperature.

Dissolution may be performed by mixing cangrelor and the solvent in oneportion or over smaller aliquots. Dissolution may also be performed overa selected period of time, for example, over 10 min to 1 h, includingover 5 min to 10 min.

When pharmaceutical formulations are being prepared, one or morepharmaceutically acceptable excipients may be added to the solvent aswell (also referred to herein as “acceptable excipient” and“excipient”). Excipients are components of a pharmaceutical formulationthat serve to maintain, stabilize or alter the physico-chemical orphysiological behavior of the active ingredient of a pharmaceuticalformulation. Suitable excipients include, but are not limited to, agentsthat modify the lyophilization behavior of the active ingredient (e.g.,cangrelor), agents that improve the rate of dissolution of the activeingredient, bulking agents and/or stabilizing agents. A bulking agentrefers to any material that fills or provides volume to the activeingredient. A stabilizing agent refers to any material which serves tominimize degradation of the active ingredient. Examples of suitableexcipients include, but are not limited to, polyols such asmonosaccharides including glucose or fructose; a disaccharide includingsucrose, maltose, or trehalose; an oligosaccharide; a polysaccharide; ora reduced sugar, such as sorbitol or mannitol. Exemplary excipientsinclude mannitol, sorbitol, sucrose, lactose, fructose and trehalose,antioxidants, buffering agents, and preservatives. Preferredpharmaceutically acceptable excipients for cangrelor are exemplified,but not limited to, those described in U.S. Pat. No. 6,114,313 and U.S.Pat. No. 6,130,208.

The cangrelor solution and one or more excipients may be efficientlymixed using methods described above.

When present, the quantity of excipient will depend on factors such asthe desired final concentration of cangrelor in the solvent, theidentity of the solvent, and the means used to remove the solvent (asdiscussed below). However, in one aspect of the invention, the amount ofexcipient included in the cangrelor solution, when present, may beadjusted to provide a cangrelor solution having a ratio of the one ormore excipients to the cangrelor of between about 5:1 and about 1:10 byweight, such as between about 3:1 and about 1:2, and about 1:2. In oneaspect, two excipients are added to the solvent, for example twopolyols, such as both sorbitol and mannitol.

The solution resulting from dissolving cangrelor in the solvent isreferred to here as the “cangrelor solution” or alternatively the “firstsolution.”

2) Preparing a pH-Adjusting Agent

The compounding process further comprises mixing a pH-adjusting agentwith the cangrelor solution to form a compounding solution. ThepH-adjusting agent may be prepared before, after, or simultaneously withthe cangrelor solution.

The pH-adjusting agent may comprise a base dissolved or mixed in asolvent, or an acid dissolved or mixed in a solvent. When thepH-adjusting agent comprises a base, the base may be neat base such as abase which is liquid at room temperature, such as triethanolamine, abase which is solid at room temperature, such as sodium hydroxide, or avolatilizable base such as ammonium carbonate.

The base may be an organic base or an inorganic base. The terms“inorganic base” and “organic base”, as used herein, refer to compoundsthat react with an acid to form a salt; compounds that produce hydroxideions in an aqueous solution (Arrhenius bases); molecules or ions thatcapture hydrogen ions (Bronsted-Lowry bases); and/or molecules or ionsthat donate an electron pair to form a chemical bond (Lewis bases). Incertain processes, the inorganic or organic base may be an alkali metalcarbonate, an alkali metal bicarbonate, an alkaline earth metalcarbonate, an alkali metal hydroxide, an alkaline earth metal hydroxide,an amine, or a phosphine. For example, the inorganic or organic base maybe an alkali metal hydroxide such as sodium hydroxide, potassiumhydroxide, cesium hydroxide, or lithium hydroxide; an alkali metalcarbonate such as potassium carbonate or sodium carbonate; or an alkalimetal bicarbonate such as sodium bicarbonate.

Solvents in which the base is dissolved or mixed may include aqueous andnon-aqueous liquids, including but not limited to, mono- and di-alcoholssuch as methanol, ethanol, isopropyl alcohol, and propylene glycol;polyhydric alcohols such as glycerol and polyethylene glycol; buffers;and water. The pH-adjusting agent may also comprise one or more carrierssuch as dissolved polyols. For instance, the sugar may be amonosaccharide such as glucose or fructose; a disaccharide such assucrose, maltose, or trehalose; an oligosaccharide; or a polysaccharide.The polyol may also be a reduced sugar, such as sorbitol or mannitol.There may be more than one carrier in the pH-adjusting agent. Thequantity of the carrier in the pH-adjusting agent may be adjusted toprovide the final product as described above.

The base is preferably mixed or dissolved in the solvent to form thepH-adjusting agent. The mixing or dissolution can be performed bymethods known in the art. For instance, the base may be added to thesolvent rapidly, slowly, in portions, at a constant rate, at a variablerate, or a combination thereof. Also, a mixing device known in the artmay be used to mix the base and the solvent. Examples of mixing devicesinclude, but are not limited to, a paddle mixer, magnetic stirrer,shaker, re-circulating pump, homogenizer, and any combination thereof.Suitable mixing rates will depend on such factors as the solvent, thedesired final concentration, and the identity of the mixing device.However, suitable mixing rates may include between about 100 and about1500 rpm, or between about 300 and about 1200 rpm. The base isadded/mixed with the solvent in a quantity that will result in apH-adjusting agent that is characterized as being between about 0.01 Nand about 5 N, which includes between about 0.1 N and 1 N. The skilledartisan will understand that the specific normality of the pH-adjustingagent will vary depending on the characteristics of the cangrelorsolution with which the pH-adjusting agent will be combined.

pH-adjusting agents are widely available and will be readily apparent tothe skilled artisan. The following are non-limiting examples: aceticacid, ammonium carbonate, ammonium phosphate, boric acid, citric acid,lactic acid, phosphoric acid, potassium citrate, potassiummetaphosphate, monobasic potassium phosphate, sodium acetate, sodiumcitrate, sodium lactate solution, dibasic sodium phosphate and monobasicsodium phosphate, sodium hydroxide, hydrochloric acid, sodiumbicarbonate, sodium carbonate, potassium bicarbonate, potassiumcarbonate, potassium hydroxide, potassium phosphate, dibasic potassiumphosphate, sodium phosphate and sodium borate.

3) Mixing the pH-Adjusting Agent with the Cangrelor Solution to Form aCompounding Solution

The pH-adjusting agent may then be mixed with the cangrelor solution toform a compounding solution (also referred to herein as a “secondsolution”). This mixing may occur by adding the pH-adjusting agent tothe cangrelor solution. Alternatively, the cangrelor solution may beadded to the pH-adjusting agent, or the pH-adjusting agent and thecangrelor solution may be added simultaneously (into a separate vessel),or there may be a combination of these addition methods. It is importantduring the adding or mixing of the pH-adjusting agent and the cangrelorsolution that pH is controlled. See below. Reference to the compoundingsolution can be a reference to the cangrelor solution during or afteraddition of the pH-adjusting agent, or it can be a reference to thepH-adjusting agent during or after addition of the cangrelor solution,or it can be a reference to the solution formed during or aftercombination of the pH-adjusting agent and the cangrelor solution.

The mixing of the pH-adjusting agent and the cangrelor solution mayoccur under controlled conditions. For example, temperature may becontrolled by means known in the art, such as by mixing the pH-adjustingagent and the cangrelor solution in a vessel inside a cooling jacket.The temperature may be set between about 1° C. and about 25° C.,including between about 2° C. and about 10° C. In some instances, thetemperature may exceed 25° C. for limited periods of time. Also, themixing of the pH-adjusting agent and the cangrelor solution may occurunder additional controlled conditions, for example such as under aninert dry gas, such as nitrogen, and/or in the absence of light.

Levels of degradants due to hydrolysis in the compounding solution areminimized by achieving and maintaining a pH of between about 7.0 andabout 9.5 in the compounding solution. Additional acceptable rangesinclude: between about 7.0 and about 8.0, between about 7.5 and about8.5, between about 8.0 and about 9.0, between about 8.5 and about 9.5,between about 7.5 and about 9.5, between about 8.0 and about 9.5,between about 7.0 and about 9.0, and between about 7.0 and about 8.5. Inparticular aspects, the pH of the compounding solution is maintained atabout pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1,8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, or 9.5.

While U.S. Pat. No. 6,114,313 teaches some limitations to the pH rangein the final formulation of cangrelor, we have found that theselimitations are insufficient to ensure the desired, low levels ofdegradants are reached. In addition, we have found that the pH rangemust be maintained throughout the process and not only in the finalformulation.

While not wishing to be bound by theory, the degradants may also begenerated by locally different pH or “hot spots,” which are defined hereas concentrated sites in the compounding solution that have muchdifferent pH levels than the surrounding environment. An example of ahot spot is a site in the compounding solution having a pH of about 12,while the surrounding solution has a pH of about 7. Degradation may alsooccur at such high pH levels in the compounding solution in general. Ithas been found that efficient and complete mixing reduces the generationof “hot spots” or high levels of pH in the compounding solution whilethe pH-adjusting agent and the cangrelor solution are being added ormixed. Thus, efficient mixing may control the overall pH level of thecompounding solution to a level not exceeding about 10.5, or a level notexceeding about 10.0, or a level not exceeding about 9.5, or even alevel not exceeding about 9.0.

Efficient mixing to minimize levels of degradation in the compoundingsolution may be achieved through various methods. One such method is toadd or combine the pH-adjusting agent and the cangrelor solutionportion-wise, i.e., in portions. For instance, the pH-adjusting agentmay be added to the cangrelor solution in portions of set quantities,wherein each addition is separated by a period of time. The quantity ofpH-adjusting agent may be approximately equal or may vary among theportions. For example, the pH-adjusting agent may be added in fourportions, wherein each portion comprises about 25% of the totalpH-adjusting agent quantity. As another example, the pH-adjusting agentmay be added in three portions, such that the first portion comprisesabout 45% of the total pH-adjusting agent quantity, the second portioncomprises about 30% of the total pH-adjusting agent quantity, and thethird portion comprises about 25% of the total pH-adjusting agentquantity.

The pH-adjusting agent may also be added in portions such that there isa combination of equal and unequal quantities. For instance, thepH-adjusting agent may be divided into four portions, wherein the firstportion comprises about 45% of the total pH-adjusting agent quantity,the second portion comprises about 25% of the total pH-adjusting agentquantity, and the third and fourth portions each comprise about 15% ofthe total pH-adjusting agent quantity.

The period of time between the additions of each portion may vary. Thisperiod may be a set duration of time regardless of the number ofportions and/or volume of the portions to be added. Alternatively, theperiod of time may vary according to the number of portions and/orvolume of the portions to be added. For example, the period of timebetween adding four equal portions may be about 5 minutes between eachaddition. As another example, the period of time after adding a firstportion comprising about 60% of the total pH-adjusting agent quantitymay be about 15 minutes, while the period of time after adding a secondportion comprising about 40% of the total pH-adjusting agent quantitymay be about 5 minutes.

The period of time between the additions of each portion may also bebased upon a set total time for adding the pH-adjusting agent. Forinstance, if the total time for adding a pH-adjusting agent is set atabout 20 minutes, then the period of time after adding each portioncomprising about 25% of the total pH-adjusting agent quantity may beabout 5 minutes. The period of time between the additions of eachportion may also be based upon a set minimal time to allow for efficientmixing so as to avoid pH “hot spots”. In certain embodiments of thepresent invention, the minimal time between the additions of twoportions of the pH-adjusting agent may be a duration of between about 5minutes and about 10 minutes, and in one example, between about 2minutes and about 5 minutes, and in another example, between about 2minutes and about 3 minutes.

Efficient mixing may also be achieved by adding the pH-adjusting agentto the cangrelor solution at a constant rate. The pH-adjusting agent maybe added at a rate of between about 0.5% and about 50% of the totalpH-adjusting agent quantity per minute; and in one example, betweenabout 1% and about 25% of the total pH-adjusting agent quantity perminute; and in another example, between about 3% and about 8% of thetotal pH-adjusting agent quantity per minute.

The pH-adjusting agent may alternatively be added at a variable rate tothe cangrelor solution. As an example, the rate may increase from about5% to about 20% of the total pH-adjusting agent quantity per minuteduring the addition of the pH-adjusting agent.

The pH-adjusting agent may also be added to the cangrelor solutionportion-wise, wherein each portion is added at a constant or variablerate. The portions may be added in equal amounts, unequal amounts, or acombination thereof. Further, each portion may be added at the same ordifferent constant rates, or the same or different variable rates, or acombination thereof. As an example, the first portion comprising 60% ofthe total pH-adjusting agent may be added at 5% of the portion volumeper minute, while four subsequent portions each comprising about 10% ofthe total pH-adjusting agent may be added at 10% of the portion volumeper minute.

Furthermore, efficient mixing may be achieved through the use of one ormore mixing devices. Examples of mixing devices include, but are notlimited to, a paddle mixer, magnetic stirrer, shaker, re-circulatingpump, homogenizer, and any combination thereof. The mixing rate of, forinstance, a paddle mixer may be between about 100 rpm and 1000 rpm, andin one example, between about 400 rpm and about 800 rpm. The mixing ratefor, as an example, a homogenizer (i.e., high shear mixing) may bebetween about 300 and about 6000 rpm, and in one example, between about1500 rpm and about 3000 rpm.

The mixing device may mix continuously during the addition of thepH-adjusting agent, or at specific periods of time, e.g., between theadditions of portions, after the pH-adjusting agent is added, etc.

In addition, more than one mixing device may be used when thepH-adjusting agent is added to the cangrelor solution. For example, apaddle mixer may be used at the surface of the cangrelor solution and ahomogenizer may be used near the bottom of the cangrelor solution. Whenmore than one mixing device is used, they may be operated at the samemixing rate or different mixing rates, or a combination thereof. Themixing devices may also be operated at the same periods of time, atdifferent periods of time, or a combination thereof, during the additionof the pH-adjusting agent. Similarly, a mixing device may be used withthe addition of the cangrelor solution to the pH-adjusting agent, orwith the addition of the pH-adjusting agent and the cangrelor solutiontogether.

Moreover, efficient mixing may be achieved through adding thepH-adjusting agent to specific sites within the cangrelor solution. Forinstance, the pH-adjusting agent may be added to the surface of thecangrelor solution or to the bottom of the cangrelor solution. In thecases wherein a mixing device is used, the pH-adjusting agent may beadded to the site of the mixing device, e.g., at the site of the paddlesof the paddle mixer or the blades of the homogenizer. The pH-adjustingagent may also be added to more than one site in the cangrelor solution;for example, the pH-adjusting agent may be added simultaneously at thetop of the cangrelor solution and at the site of the mixing device.Alternatively, the cangrelor solution may be added to the pH-adjustingagent at specific sites and at more than one site within thepH-adjusting agent, as described above.

Optionally, once the compounding solution is formed, the pH or the finalvolume of the compounding solution may be adjusted to the target levelbefore removal of the solvent (see below). The pH or volume can beadjusted using methods known in the art, for instance, the addition ofadditional solvent or pH-adjusting agent as described above.

When pharmaceutical formulations are being prepared, one or morepharmaceutically acceptable excipients may be added to the compoundingsolution. Such additions may be in place of the addition of excipientsdescribed above during production of a cangrelor solution, or inaddition to the addition of excipients described above during productionof a cangrelor solution. Thus, excipients can be added during productionof the cangrelor solution, during production of the compoundingsolution, or both.

The timing and manner in which the excipients are added to thecompounding solution is not critical. Thus, for example, the excipientsmay be added to the compounding solution before or after thepH-adjusting agent is added, or added during some or all of the periodover which the pH-adjusting agent is added. Similarly, the compoundingsolution may be added to the excipient. Suitable excipients include anagent modifying the lyophilization behavior of the active pharmaceuticalingredient, an agent improving the rate of dissolution of the activepharmaceutical ingredient, a bulking agent or as a stabilizing agent. Insome embodiments, the excipients may be polyols. For example, the polyolmay be a monosaccharide such as glucose or fructose; a disaccharide suchas sucrose, maltose, or trehalose; an oligosaccharide; or apolysaccharide. Alternatively, the polyol may be a reduced sugar, suchas sorbitol or mannitol. The compounding solution and one or moreexcipients may be efficiently mixed using methods described above. ThepH of the resulting solution may be checked and if it is found to beoutside the desired range of between about pH 7.0 and about pH 9.5,additional pH-adjusting agent may be added to minimize generation ofdegradants.

When present, the quantity of excipient will depend on factors such asthe desired final concentration of cangrelor in the compoundingsolution, the identity of the solvents, and the means used to remove thesolvents (as discussed below). However, in one aspect of the invention,the compounding solution may be adjusted to provide a pharmaceuticalformulation having a ratio of the one or more excipients to thecangrelor of between about 5:1 and about 1:10 by weight, such as betweenabout 3:1 and about 1:2, and about 1:2. In one aspect, two excipientsare added to the compounding solution, for example two polyols, such asboth sorbitol and mannitol. Stated in another fashion, thepharmaceutical formulations of the invention comprise high puritycangrelor, expressed as the free acid but present as the free acid or asalt thereof, in a range of about 10-30% and one or morepharmaceutically acceptable excipients in a range of about 90-70%, byweight of the pharmaceutical formulation. In one aspect, thepharmaceutical formulations of the invention comprise high puritycangrelor, expressed as the free acid but present as the free acid or asalt thereof, in a range of about 15-25% and one or morepharmaceutically acceptable excipients in a range of about 85-75%, byweight. In another aspect, the pharmaceutical formulations of theinvention comprise high purity cangrelor, expressed as the free acid butpresent as the free acid or a salt thereof, in a range of about 16-22%and one or more pharmaceutically acceptable excipients in a range ofabout 84-78%, by weight. In a further aspect, the pharmaceuticalformulations of the invention comprise high purity cangrelor, expressedas the free acid but present as the free acid or a salt thereof, in arange of about 16-21% and one or more pharmaceutically acceptableexcipients in a range of about 84-79%, by weight. In certain aspects,the amount of high purity cangrelor, expressed as the free acid, in apharmaceutical formulation is not more than about 30%, 29%, 28%, 27%,26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%, by weight of theformulation, with the remainder of the weight comprising the one or morepharmaceutically acceptable excipients, moisture and counterions.

When an excipient is added to the compounding solution, the pH or thevolume of the solution may be determined after addition and, if needed,adjusted to the target level before removal of the solvent (see below).The pH or volume can be adjusted using methods known in the art, forinstance, the addition of additional solvent or pH-adjusting agent asdescribed above.

The compounding solution may also be sterilized before the removal ofsolvent. The compounding solution may undergo aseptic filtration using,for example, a membrane filter, such as 0.2 μm membrane filter, tosterilize the compounding solution. Techniques of sterilizing thecompounding solution are known in the art (see, e.g., Berovic, M.Biotechnol. Annu. Rev. 2005, 11, 257-279, incorporated herein byreference in its entirety).

When the compounding solution is sterilized, the pH or the volume of theresulting solution may be determined after sterilization and, if needed,adjusted to the target level before removal of the solvent (see below).The pH or volume can be adjusted using methods known in the art, forinstance, the addition of additional solvent or pH-adjusting agent asdescribed above.

Furthermore, following sterilization, the compounding solution may bealiquoted into containers such as vials, bottles, ampoules, syringes,etc.

4) Removal of Solvent from the Compounding Solution

The compounding process further comprises removing solvents from thecompounding solution.

Solvent removal from the compounding solution may be achieved throughlyophilization, which comprises freezing the compounding solution andthen reducing the surrounding pressure to allow the frozensolvent/moisture in the material to sublime directly from a solid phaseto a gas phase. The lyophilization process may be performed by methodsknown in the art (see, e.g., Liu, J. Pharm. Dev. Technol. 2006, 11,3-28; Tang, X.; Pikal, M. J. Pharm. Res. 2004, 21, 191-200; Nail, S. L.;Jiang, S.; Chongprasert, S.; Knopp, S. A. Pharm. Biotechnol. 2002, 14:281-360; U.S. Pat. No. 7,351,431, and U.S. Pat. No. 6,821,515; each ofwhich is incorporated herein by reference in its entirety).

Solvents may also be removed from the compounding solution through othertechniques such as spray drying and spray-freeze drying (see, e.g., Lee,G. Pharm. Biotechnol. 2002, 13, 135-58; Maa, Y.-F.; Prestrelski, S. J.Curr. Pharm. Biotechnol. 2000, 1, 283-302; each of which is incorporatedherein by reference in its entirety), vacuum drying, super criticalfluid processing, air drying, or other forms of evaporative drying, asknown in the art.

Lyophilization represents a process which generally comprises the stepsof (a) chilling a solution to a temperature from about 5° C. to about−80° C., wherein the temperature is maintained for at least about 20minutes to about 4 hours, (b) freezing the solution to a temperature offrom about 0° C. to about −80° C., to produce a frozen mixture, whereinthe temperature is maintained for at least about 30 minutes to about 20hours, and (c) subjecting the frozen mixture to a primary drying stage,which comprises applying a vacuum to reduce the pressure by an amounteffective to remove aqueous solvent from the frozen mixture, and, whileapplying the vacuum, changing the temperature of the frozen mixture to aprimary drying temperature, wherein the primary drying temperature isfrom about 0° C. to about −50° C., and wherein the primary dryingtemperature is maintained for at least about 10 hours to about 50 hours.

Lyophilization may be performed over several steps, for example byconducting a step at a temperature range of between about −15° C. andabout −50° C. and a pressure of between about 0.05 torr and about 0.5torr and conducting a second step at a temperature range of betweenabout −10° C. and about −20° C. and a pressure of between about 0.1 torrand about 0.5 torr. In other instances, only one lyophilization step maybe required.

For example, the compounding solution may be frozen using suchtechniques as, but not limited to, mechanical refrigeration, dry ice,and liquid nitrogen. The temperature may be cooled to a range of betweenabout 0° C. and about −80° C., and in one example, between about −10° C.and about −35° C. The primary lyophilization step may be characterizedby a lowered pressure of between about 0.05 torr and about 10 torr, andin one example, between about 0.1 torr and about 1 torr. The secondarylyophilization step may be characterized by a pressure between about0.05 torr and about 5 torr, and in one example, between about 0.1 torrand about 1 torr. In other instances, only one lyophilization step maybe required.

In some instances, further drying may be performed after the bulk of thesolvent was removed for example by maintaining the material at atemperature range of about 10° C. and 45° C. and a reduced pressure ofbetween 0.05 torr and 5 torr, and in one example, at a temperature rangeof about 20° C. and 40° C. and a reduced pressure of between 0.1 torrand 1 torr. This additional drying step may be performed for a durationof between about 1 hour and about 10 hours, and in one example, betweenabout 3 hours and about 6 hours

In certain embodiments of the invention, removal of the solvent iseffected under conditions where the residual moisture in the high puritycangrelor or salt thereof, and in a pharmaceutical formulationcomprising high purity cangrelor, or a salt thereof, as an activeingredient, is less than about 2.0% on a weight basis to minimize thegeneration of degradants during further processing and storage. In otherembodiments of the invention, the removal of the solvent will result inhigh purity cangrelor or salt thereof, and in a pharmaceuticalformulation comprising high purity cangrelor, or a salt thereof, as anactive ingredient, with less than about 2.0% moisture on a weight basisand a pH of between about 7.0 and about 9.5 to minimize the generationof degradants during further processing and storage. In aspects of theseembodiments, the residual moisture is less than about 3.4%, 3.3%, 3.2%,3.1%, 3.0%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2.0%,1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%,0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% on a weight basis.

The proper combinations of temperatures, reduced pressures and durationsof the processes used for solvent removal are critical in order tominimize the levels of degradants generated during the processes, andupon storage of the high purity cangrelor or salt thereof and apharmaceutical formulation comprising high purity cangrelor or a saltthereof.

A suitable process according to the invention is a vial freeze-dryingprocess. Such a process comprises filling sterile vials with a sterilefiltered solution of the composition according to the invention, such asa compounding solution. A sterile freeze-drying stopper is partiallyinserted into the vial which is frozen, e.g. at a temperature from −30to −40° C., and thereafter vacuum dried in the frozen state. Afterdrying, the stopper is fully inserted before removing the vial from thelyophilization unit.

It is possible that during solvent removal, the pH of the resultingmaterial is altered, either as a result of the concentration of the baseor as a result of the removal of a volatile base. The selected processmust ensure that the pH of the compounding solution or of the resultingmaterial remains in the range of about 7.0 to about 9.5.

The presence of degradants will increase over time as the compoundingsolution is stored or manipulated before the removal of solvent.Therefore the length of time between the dissolution of cangrelor toform the first solution and the removal of the solvent must be kept to aminimum to minimize the levels of the degradants generated. For example,this length of time should not exceed about 48 hours, and in aspects,not exceed about 36 hours, about 30 hours, about 24 hours, about 20hours, about 16 hours, about 12 hours, about 8 hours, or about 4 hours.

To prevent oxidative processes brought about by the presence of oxygenand hydrolytic processes brought about by the presence of water, uponcompletion of the removal of the solvent, the resulting materialobtained can be stored in an environment made of a chemically inert andmoisture free gas within the storage vessel. This chemically inert andmoisture free gas may be nitrogen or argon. In particular, thechemically inert dry gas can be introduced upon release of the vacuum atthe end of lyophilization or vacuum drying cycles.

In aspects of the invention disclosed herein, the level of impurity Apresent in the high purity cangrelor or salt thereof, or inpharmaceutical formulations comprising the high purity cangrelor or saltthereof, is less than about 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7,0.6, 0.5, 0.4, 0.3, 0.2 or 0.1% by weight of the drug product.

In aspects of the invention disclosed herein, the level of impurity Bpresent in the high purity cangrelor or salt thereof, or inpharmaceutical formulations comprising the high purity cangrelor or saltthereof, is less than about 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2or 0.1% by weight of the drug product.

In aspects of the invention disclosed herein, the level of impurity Cpresent in the high purity cangrelor or salt thereof, or inpharmaceutical formulations comprising the high purity cangrelor or saltthereof, is less than about 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2or 0.1% by weight of the drug product.

In aspects of the invention disclosed herein, the level of impurity Dpresent in the high purity cangrelor or salt thereof, or inpharmaceutical formulations comprising the high purity cangrelor or saltthereof, is less than about 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2or 0.1% by weight of the drug product.

In aspects of the invention disclosed herein, the level of impurity Epresent in the high purity cangrelor or salt thereof, or inpharmaceutical formulations comprising the high purity cangrelor or saltthereof, is less than about 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2or 0.1% by weight of the drug product.

In aspects of the invention disclosed herein, the combined level ofimpurities A and D present in the high purity cangrelor or salt thereof,or in pharmaceutical formulations comprising the high purity cangreloror salt thereof, is less than about 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9,1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5,0.4, 0.3, 0.2 or 0.1% by weight of the drug product.

In aspects of the invention disclosed herein, the level of impurities Aand D present in the high purity cangrelor or salt thereof, or inpharmaceutical formulations comprising the high purity cangrelor or saltthereof, is each less than about 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8,1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4,0.3, 0.2 or 0.1% by weight of the drug product.

In aspects of the invention disclosed herein, the combined level ofimpurities A, B, C, D and E present in the high purity cangrelor or saltthereof, or in pharmaceutical formulations comprising the high puritycangrelor or salt thereof, is less than about 5.0, 4.5, 4.0, 3.5, 3.0,2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2,1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1% by weight ofthe drug product. The particular impurities in the combined amount mayvary in their individual concentrations or be about the same. Further,the skilled artisan will understand that any combination of the fiveimpurities A, B, C, D and E, in any amount, may be present in the highpurity cangrelor or salt thereof, although with a combined total of lessthan about 5.0% by weight of the drug product, and less than about 1.5%in some aspects. For example, there may be only one, or two, or three,or four, or all five of the impurities present in the high puritycangrelor or salt thereof.

5) Filling in Storage Vessels

The dried high purity cangrelor and pharmaceutical formulationscomprising high purity cangrelor should be stored in a vessel that willprevent exposure of the drug product or formulations to moisture. Insome aspects, exposure of the drug product or formulations to light mayalso be blocked. In a suitable example, the drug product andformulations are stored in sealed vessels such as stoppered vials.Filling of these vessels may be concomitant with solvent removal. Thatis, the compounding solution is loaded into the vessel and the solutionis dried in the vessel as described above.

By methods known to those skilled in the art, vessels and their stoppersused for storing drug products and pharmaceutical formulations arewashed, sterilized and dried prior to use. Residual moisture in vesselsand their stoppers following this process can be transferred to the drugproduct and formulations over time and result in the appearance ofdegradants produced through hydrolytic process. Therefore, care shouldbe taken to minimize the amount of residual moisture in the vessels andtheir stoppers.

The vessels must also be sealed sufficiently to ensure that oxygen andmoisture do not penetrate over time, thereby minimizing the levels ofdegradants formed due to oxidative or hydrolytic processes. The vesselsmay be sealed by a stopper held in place by sleeves, by crimps or byoverseals. The stoppers may be made from an elastic material such asrubber and the sleeves or crimps may be made from a malleable metal suchas aluminum. The appropriateness of the seal can be checked by methodsknown to those skilled in the art, such as through helium leak detection(see, e.g., Kirsch, L. E.; Nguyen, L.; Moeckly, C. S. PDA J Pharm SciTechnol. 1997, 51, 187-194, the disclosure of which is herebyincorporated by reference in its entirety). For example, the helium leakrate may be between about 1×10⁻⁶ std.cc/sec and about 1×10⁻⁴ std.cc/sec.

The components of the vessel, such as the stopper, that are made ofelastic materials may be selected for their ability to absorb as littlemoisture as possible during washing and sterilization. These componentsmay be made of butyl rubber.

Prior to use, the vessel and the stopper are dried at a sufficienttemperature and for a sufficient duration to ensure that they transferas little moisture as possible to the dried drug products andpharmaceutical formulations. For example, they may be dried at atemperature of about 70° C. to about 150° C. for a duration of about 1hour to about 24 hours, such as about 1 hour to 4 hours.

In an embodiment of the invention, the sealed vessel and its componentsare selected and dried so that the amount of moisture found in the highpurity cangrelor and pharmaceutical formulations comprising high puritycangrelor remains below 5.0% on a weight basis, and below 2.0% in someaspects, over a period of at least about 24 months. In particularaspects, the amount of moisture found in the high purity cangrelor andpharmaceutical formulations remains below 4.5, 4.0, 3.5, 3.0, 2.5, 2.4,2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 or0.5% on a weight basis over a period of at least about 3, 6, 9, 12, 15,18, 21, 24, 36 or 48 months. The present invention also encompasses highpurity cangrelor and pharmaceutical formulations defined in this manner.

In another embodiment of the invention, the sealed vessel and itscomponents are selected and dried so that after a period of about 12months, the high purity cangrelor and pharmaceutical formulationscomprising high purity cangrelor are characterized by a pH of betweenabout 7.0 and 9.5 for a 1% solution by weight, an amount of moistureless than about 5% on a weight basis (less than about 2.0% in someaspects), a maximum level of the impurities A, B, C and D not exceedingabout 1% each by weight of the drug product (not exceeding 0.5% in someaspects) and a maximum level of impurity E not exceeding about 0.5% byweight of the drug product. The present invention also encompasses highpurity cangrelor and pharmaceutical formulations defined in this manner.

In another embodiment of the invention, the sealed vessel and itscomponents are selected and dried so that after a period of about 12months, the high purity cangrelor and pharmaceutical formulationscomprising high purity cangrelor are characterized by a pH of betweenabout 7.0 and 9.5 for a 1% solution by weight, an amount of moistureless than about 5% on a weight basis (less than about 2.0% in someaspects), and a maximum level of impurity A not exceeding about 1% byweight of the drug product (not exceeding about 0.5% in some aspects), amaximum level of impurity B not exceeding about 0.5% by weight of thedrug product (not exceeding about 0.2% in some aspects), a maximum levelof impurity C not exceeding about 0.3% by weight of the drug product, amaximum level of impurity D not exceeding about 0.2% by weight of thedrug product and a maximum level of impurity E not exceeding about 0.5%by weight of the drug product. The present invention also encompasseshigh purity cangrelor and pharmaceutical formulations defined in thismanner.

In another embodiment of the invention, the sealed vessel and itscomponents are selected and dried so that after a period of about 12months, the high purity cangrelor and pharmaceutical formulationscomprising high purity cangrelor are characterized by a pH of betweenabout 7.0 and 9.5 for a 1% solution by weight, an amount of moistureless than about 5% on a weight basis (less than about 2.0% in someaspects), and a maximum combined level of impurities A, B, C, D and Enot exceeding about 5.0% by weight of the drug product, or a maximumcombined level of impurities A, B, C, D and E not exceeding about 2.0%by weight of the drug product, or a maximum combined level of impuritiesA, B, C, D and E not exceeding about 1.5% by weight of the drug product,or a maximum combined level of impurities A, B, C, D and E not exceedingabout 1.3% by weight of the drug product. The present invention alsoencompasses high purity cangrelor and pharmaceutical formulationsdefined in this manner.

Formulations

The high purity cangrelor and pharmaceutical formulations of the presentinvention may be used in methods of inhibiting platelet activation andaggregation in vitro, in vivo and ex vivo. Such methods form the basisof therapeutic methods in animals such as humans. Providing high puritycangrelor and pharmaceutical formulations comprising high puritycangrelor in vessels, as discussed herein, will greatly aid in thepractice of such methods.

The amount of high purity cangrelor or a pharmaceutical formulationcomprising high purity cangrelor included in a vessel, such as astoppered vial, will depend on the manner in which the drug product orformulation will be used. The amount may be one that allows the drugproduct or formulation to be reconstituted in the vessel and then usedin vitro or ex vivo, or administered to a subject, without furtherdilution. Alternatively, the amount may be one that requires the drugproduct or formulation to be further diluted after reconstitution in thevessel and prior to use.

As an example, high purity cangrelor or a pharmaceutical formulationcomprising the drug product may be supplied in single-use vials. Eachsingle-use vial may contain about 50 mg of drug product or theformulation. When reconstituted with a sterile aqueous solution, areconstituted solution with a pH of about 8-9.5 results. Reconstitutionmay be performed using water for injection, 0.9% NaCl, buffered saline,dextrose (e.g., 5% dextrose in water) or water as the sterile aqueoussolution.

In some aspects, the pharmaceutical formulations of the presentinvention can be characterized by the amount of time required toreconstitute the formulations when mixed with a sterile aqueoussolution. The reconstitution time, i.e., time required to put thepharmaceutical formulations in solution, may be characterized as notexceeding about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute.

Reconstitution times may be determined, for example, by adding aselected level of sterile aqueous solution to a unit dosage vialcomprising the cangrelor pharmaceutical formulation. Immediately afteradding the appropriate solution (e.g., water, water for injection,saline, etc.), a timer is started. The vial is shaken vigorously, withinversion, for approximately 10 seconds. The vial is viewed to determineif the solid has dissolved. If the solid has not completely dissolved,the vial is shaken for another 10 seconds. These steps are repeateduntil all the solid dissolves, at which point the time is stopped andrecorded.

When used in the treatment of a subject, the reconstituted formulationmay be administered to a subject via parenteral modes of administration,including without limitation, intradermal, subcutaneous (s.c., s.q.,sub-Q, Hypo), intramuscular (i.m.), intravenous (i.v.), intraperitoneal(i.p.), intra-arterial, intramedullary, intracardiac, intraspinal, andintrathecal (spinal fluids) modes. Any known device useful forparenteral injection or infusion of drug formulations can be used toeffect such administration. In noted aspects and embodiments of thepresent invention, administration of the pharmaceutical compositions isvia parenteral administration, preferably intravenous administration.

In intravenous (IV) administration, a sterile reconstituted formulationcan be diluted in any of the commonly used intravenous fluids andadministered by infusion. Intravenous fluids include, withoutlimitation, physiological saline, 0.9% NaCl, phosphate buffered saline,5% dextrose in water, 0.002% polysorbate 80 (Tween-80™) in water orRinger's™ solution.

In intramuscular preparations, a sterile reconstituted formulation canbe diluted and administered in a pharmaceutical diluent such asWater-for-Injection (WFI), physiological saline, 0.9% NaCl or 5%dextrose in water.

Suitable final concentrations of high purity cangrelor, or salt thereof,in the reconstituted formulations will vary depending on the particularuse to which the formulation will be put, but may include high puritycangrelor, or salt thereof, at a concentration of about 0.1, 0.5, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 mg/mL in 0.9% NaCl, or a concentration ofabout 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/mL in 5% dextrose.

Dosage

As used herein, the terms “dose”, “dosage”, “unit dose”, “unit dosage”,“effective dose”, “effective amount” and related terms refer tophysically discrete units that contain a predetermined quantity of highpurity cangrelor, or salt thereof, calculated to produce a desiredtherapeutic effect. These terms are synonymous with the therapeuticallyeffective amounts and amounts sufficient to achieve the stated goals ofthe methods disclosed herein.

Particular doses of the pharmaceutical formulations of the presentinvention will vary depending upon the stated goals of the methods(treating, preventing or reducing), the physical characteristics of thesubject, existence of related or unrelated medical conditions, thecomposition of the formulation and the means used to administer the drugproduct to the subject. The specific dose for a given subject willgenerally be set by the judgment of the attending physician.

When administered as an intravenous (IV) formulation, a pharmaceuticalformulation comprising high purity cangrelor, or salt thereof, may beadministered as a bolus, as a continuous infusion, or as a bolusfollowed by a continuous infusion. When administered as a bolus, a doseof about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95 or 100 μg/kg cangrelor, or more, is administered to thesubject. In preferred embodiments, between about 20 and 40 μg/kgcangrelor is administered, more preferably about 30 μg/kg. Whenadministered as a continuous infusion, cangrelor may be administered atabout 0.1, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30μg/kg/min, or more, to the subject. In preferred embodiments, betweenabout 0.1 and 10 μg/kg/min cangrelor is administered, more preferablyabout 4 μg/kg/min. The skilled artisan will understand that differentdosages may be administered during different points of a medicalprocedure. Thus the dosages may differ in the periods before, during andafter a medical procedure.

In each of the embodiments where the pharmaceutical formulation isadministered as continuous intravenous infusion, the infusion maycontinue for at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100,120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300,320, 340 or 360 minutes, or more. The skilled artisan will understandthat the period of time over which the pharmaceutical formulation isadministered may be shorter or longer than the indicated times due tothe particular characteristics of a subject.

Where the pharmaceutical formulation is administered in conjunction withthe implantation of a stent, such as during PCI, the bolus may beadministered within about 360, 300, 240, 180, 120, 90, 60, 30 or 15minutes prior to the beginning of the procedure.

In addition to the pharmaceutical formulations of the present inventioncomprising cangrelor, the skilled artisan will understand that one, two,three, four, five or more additional anti-thrombotic agents may be usedin combination with cangrelor, such as bivalirudin. As a furtherexample, aspirin (100-500 mg daily) may be administered in conjunctionwith the pharmaceutical formulations.

Using High Purity Cangrelor and Pharmaceutical Formulations

As indicated above, the high purity cangrelor and pharmaceuticalformulations of the present invention may be used in methods ofinhibiting platelet activation and aggregation in vitro, in vivo and exvivo.

While not intending to be limited by doing so, the following areexamples of particular methods that may be practiced using the highpurity cangrelor or the pharmaceutical formulations of the presentinvention and are thus further embodiments of the invention.

In a general aspect, the present invention includes methods ofinhibiting platelet activation, aggregation, or both, in a subject,comprising administering an effective amount of a pharmaceuticalformulation of the present invention to a subject in need thereof,thereby inhibiting platelet activation, aggregation, or both, in asubject. The subject may be undergoing percutaneous coronaryintervention (PCI) or another catherization technique. The subject maybe undergoing treatment for acute coronary syndromes (ACS), or aclotting disorder in general.

In related embodiments, the present invention includes methods ofinhibiting platelet granule release in a subject, comprisingadministering an effective amount of a pharmaceutical formulation of thepresent invention to a subject in need thereof, thereby inhibitingplatelet granule release in a subject. The invention includes methods ofinhibiting platelet-leukocyte aggregation in a subject, comprisingadministering an effective amount of a pharmaceutical formulation of thepresent invention to a subject in need thereof, thereby inhibitingplatelet-leukocyte aggregation in a subject. The invention includesmethods of inhibiting platelet-granulocyte aggregation in a subject,comprising administering an effective amount of a pharmaceuticalformulation of the present invention to a subject in need thereof,thereby inhibiting platelet-granulocyte aggregation in a subject. Theinvention includes methods of inhibiting platelet loss from the blood ofa subject, comprising administering an effective amount of apharmaceutical formulation of the present invention to a subject in needthereof, thereby inhibiting platelet loss from the blood of a subject.

In a related aspect, the present invention includes methods ofinhibiting platelet activation, aggregation, or both, comprisingcontacting platelets with an effective amount of a high puritycangrelor, or a salt thereof, thereby inhibiting platelet activation,aggregation, or both. The method may be practiced in vitro, in vivo orex vivo.

In related embodiments, the present invention includes methods ofinhibiting platelet granule release, comprising contacting plateletswith an effective amount of a high purity cangrelor, or a salt thereof,thereby inhibiting platelet granule release. The invention includesmethods of inhibiting platelet-leukocyte aggregation, comprisingcontacting platelets with an effective amount of a high puritycangrelor, or a salt thereof, thereby inhibiting platelet-leukocyteaggregation. The invention includes methods of inhibitingplatelet-granulocyte aggregation, comprising contacting platelets withan effective amount of a high purity cangrelor, or a salt thereof,thereby inhibiting platelet-granulocyte aggregation. The inventionincludes methods of inhibiting platelet loss from the blood, comprisingcontacting platelets with an effective amount of a high puritycangrelor, or a salt thereof, thereby inhibiting platelet loss from theblood. The methods may be practiced in vitro, in vivo or ex vivo.

The pharmaceutical formulations of the present invention may be used inany disease, condition or procedure in a subject where plateletaggregation is involved. The pharmaceutical formulations of the presentinvention may thus act as anti-thrombotic agents and they are indicatedin the treatment or prevention of diseases and conditions including, butnot limited to, stent thrombosis, myocardial infarction, thromboembolicstroke and peripheral vascular disease. They are also indicated for usein reducing mortality in a subject undergoing stent thrombosis orexperiencing myocardial infarction. They are further indicated in thetreatment or prevention of the sequelae of thrombotic complications fromangioplasty, stent implantation, thrombolysis, endarterectomy, coronaryand vascular graft surgery, renal dialysis and cardio-pulmonary bypass.Additional indications include the treatment or prevention ofdisseminated intravascular coagulation, deep vein thrombosis,pre-eclampsia/eclampsia, tissue salvage following surgical or accidentaltrauma, vasculitis, arteritis, thrombocythaemia, ischemia and migraine.

The pharmaceutical formulations of the present invention are alsoindicated procedures such as percutaneous coronary intervention (PCI)and coronary artery bypass graft (CABG) surgery.

The present invention thus includes methods of protecting plateletfunction during medical procedures. Such medical procedures include oneor more of extracorporeal circulation (ECC) and hypothermia. The methodscomprise administering an effective amount of a pharmaceuticalformulation of the present invention to a subject undergoing a medicalprocedure that includes ECC or hypothermia, or both. In embodiments ofthe methods, the invention is directed to methods of protectingplatelets in the blood of a subject undergoing an ECC-based medicalprocedure, a hypothermia-based medical procedure or a hypothermicECC-based medical procedure, where the method comprises administering aneffective amount of a pharmaceutical formulation of the presentinvention to a subject undergoing such a procedure, thereby protectingplatelets in the blood of the subject. The protection of plateletsthrough these methods includes, but is not limited to, inhibitingactivation of platelets, inhibiting platelet granule release, inhibitingplatelet-leukocyte aggregation (including platelet-granulocyteaggregation), inhibiting platelet aggregation and inhibiting plateletloss from the blood of the subject.

Thus, in one embodiment the method inhibits activation of platelets inthe blood of a subject undergoing an ECC-based medical procedure, ahypothermia-based medical procedure, or a hypothermic ECC-based medicalprocedure, wherein the method comprises administering an effectiveamount of a pharmaceutical formulation of the present invention to asubject undergoing such a procedure, thereby inhibiting activation ofplatelets in the blood of the subject.

A second embodiment the method inhibits platelet granule release in theblood of a subject undergoing an ECC-based medical procedure, ahypothermia-based medical procedure, or a hypothermic ECC-based medicalprocedure, and comprises administering an effective amount of apharmaceutical formulation of the present invention to a subjectundergoing such a procedure.

In a third embodiment the method inhibits platelet-leukocyte aggregationin the blood of a subject undergoing an ECC-based medical procedure, ahypothermia-based medical procedure, or a hypothermic ECC-based medicalprocedure, and comprises administering an effective amount of apharmaceutical formulation of the present invention to a subjectundergoing such a procedure. In one aspect, the platelet-leukocyteaggregation is platelet-granulocyte aggregation.

In a fourth embodiment the method inhibits platelet loss from the bloodof a subject undergoing an ECC-based medical procedure, ahypothermia-based medical procedure, or a hypothermic ECC-based medicalprocedure, and comprises administering an effective amount of apharmaceutical formulation of the present invention to a subjectundergoing such a procedure.

The present invention includes methods of treating stent thrombosis. Thecourse of treatment will generally follow implantation of a stent into asubject, where the subject is suspected of having or known to havedeveloped a thrombus associated with a stent. The pharmaceuticalformulation comprising cangrelor may be a bolus intravenous dosage formor a continuous intravenous infusion dosage form, and may beadministered in combination with an oral dosage form. The course oftreatment may last for a period of hours, days, weeks, months or years.The pharmaceutical formulation comprising cangrelor may thus beadministered to a subject to treat stent thrombosis for about 1, 2, 3,4, 5, 6, or 7 days, for about 1, 2, 3 or 4 weeks, or for about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 or more months, after the implantation of avascular stent or after a diagnosis of stent thrombosis. In particularaspects, the pharmaceutical formulation may be administered to thesubject as an intravenous bolus, as a continuous intravenous infusion,as an intravenous bolus followed by continuous intravenous infusion, orsome combination thereof, and optionally, in combination with an oraldosage form. In a particular example, the pharmaceutical formulation isadministered to the subject in a continuous intravenous infusion dosageform over a period of at least about 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4hours, or more. The methods of treatment of the present inventioninclude methods wherein the pharmaceutical formulation is administeredto the subject beginning about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 months, or more, after stent implantation. The treatmentmay be once, twice, thrice or more times a day, once every two days,once every three days, once every four days, once every five days, onceevery six days, once a week, once every 10 days, once every two weeks,once every three weeks, once a month, or even less frequently.

The present invention includes methods of preventing stent thrombosis orreducing mortality in a subject undergoing stent implantation. Thecourse of prevention will generally be associated with a medicalprocedure in which a stent is being implanted into the subject. Thecourse of treatment may be limited to the administration of thepharmaceutical formulation prior to the beginning of the procedure,during the procedure or after the procedure. Alternatively, the courseof treatment may comprise administering the pharmaceutical formulationprior to the procedure and during the procedure, or during the procedureand after the procedure, or prior to the procedure and after theprocedure. The skilled artisan will also understand that the course oftreatment may begin prior to the procedure and continue until some pointafter the completion of the procedure. The skilled artisan willunderstand that the pharmaceutical formulation may be administered tothe subject via different dosage forms, such as via intravenous infusionduring the procedure and an oral dosage form for a number of days ormonths after the procedure has been completed.

When administered before stent implantation, the pharmaceuticalformulation is preferably administered to the subject in an oral dosageform, a bolus intravenous dosage form, a continuous intravenous infusiondosage form, or as an intravenous bolus followed continuous intravenousinfusion, within about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7 or 7.5 hours, or more, prior to stent implantation. Whenadministered as a continuous intravenous infusion dosage form, thepharmaceutical composition is preferably administered to the subject asa continuous intravenous infusion over about a 0.5, 1, 1.5, 2, 2.5, 3,3.5 or 4 hour period, or more.

When administered during stent implantation, the pharmaceuticalformulation is preferably administered to the subject in an oral dosageform, a bolus intravenous dosage form, a continuous intravenous infusiondosage form, or as an intravenous bolus followed continuous intravenousinfusion. When administered as a continuous intravenous infusion, theinfusion may continue over about a 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4 hourperiod, or more. The continuous intravenous infusion may also simplylast for the duration of the procedure.

When administered after stent implantation, the pharmaceuticalformulation is preferably administered to the subject in an oral dosageform, a bolus intravenous dosage form, a continuous intravenous infusiondosage form, or as an intravenous bolus followed continuous intravenousinfusion, for a period of about 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5 or 7 hours, or more, after the completion of theprocedure. When administered as a continuous intravenous infusion, theinfusion may continue over about a 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4 hourperiod, or more.

When administered both before and during the procedure, thepharmaceutical formulation may be administered to the subject in an oraldosage form, a bolus intravenous dosage form, a continuous intravenousinfusion dosage form, or as an intravenous bolus followed continuousintravenous infusion, within about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7 or 7.5 hours, or more, prior to stent implantation,and administered to the subject in an oral dosage form, a bolusintravenous dosage form, a continuous intravenous infusion dosage form,or as an intravenous bolus followed continuous intravenous infusion,during the procedure. When administered as a continuous intravenousinfusion, the infusion may continue over about a 0.5, 1, 1.5, 2, 2.5, 3,3.5 or 4 hour period, or more. The continuous intravenous infusion mayalso simply last for the duration of the procedure.

When administered during and after the procedure, the pharmaceuticalformulation may be administered to the subject in an oral dosage form, abolus intravenous dosage form, a continuous intravenous infusion dosageform, or as an intravenous bolus followed continuous intravenousinfusion, and administered to the subject in an oral dosage form, abolus intravenous dosage form, a continuous intravenous infusion dosageform, or as an intravenous bolus followed continuous intravenousinfusion, for a period of about 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5 or 7 hours, or more, after the completion of theprocedure. When administered as a continuous intravenous infusion, theinfusion may continue over about a 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4 hourperiod, or more.

When administered both before and after the procedure, thepharmaceutical formulation may be administered to the subject in an oraldosage form, a bolus intravenous dosage form, a continuous intravenousinfusion dosage form, or as an intravenous bolus followed continuousintravenous infusion, within about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7 or 7.5 hours, or more, prior to stent implantation,and administered to the subject in an oral dosage form, a bolusintravenous dosage form, a continuous intravenous infusion dosage form,or as an intravenous bolus followed continuous intravenous infusion, fora period of about 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5 or 7 hours, or more, after the completion of the procedure.When administered as a continuous intravenous infusion, the infusion maycontinue over about a 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4 hour period, ormore.

When administered before, during and after the procedure, thepharmaceutical formulation may be administered to the subject (i) in anoral dosage form, a bolus intravenous dosage form, a continuousintravenous infusion dosage form, or as an intravenous bolus followedcontinuous intravenous infusion, within about 0.5, 1, 1.5, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 or 7.5 hours, or more, prior to theprocedure, (ii) in an oral dosage form, a bolus intravenous dosage form,a continuous intravenous infusion dosage form, or as an intravenousbolus followed continuous intravenous infusion, during the procedure,and (iii) in an oral dosage form, a bolus intravenous dosage form, acontinuous intravenous infusion dosage form, or as an intravenous bolusfollowed continuous intravenous infusion, for a period of about 0.25,0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 or 7 hours, ormore, after the completion of the procedure. When the dosage form iscontinuous intravenous infusion, the infusion may continue over about a0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4 hour period, or more.

In the methods of the invention directed to methods of reducingmortality in a subject undergoing stent implantation, mortality may bereduced within a period of about 24, 36 or 48 hours after stentimplantation, within a period of about 30 days after stent implantation,within a period of about six months after stent implantation, or withina period of about one year after stent implantation. In preferredembodiments, mortality is reduced by at least about 0.2%, 0.4%, 0.6%,0.8%, 1.0% or 1.2% during the period in comparison to a subject notreceiving cangrelor.

Stent thrombosis may result from any means related to the implantation,presence, or maintenance of a stent in the vasculature of a subject. Forexample, stent thrombosis may be induced by implantation of a stent,such as bare-metal stent or a drug-eluting stent, into a subject.Similarly, stent thrombosis may develop over time due to the presence ofa stent, such as a bare-metal stent or a drug-eluting stent, in thesubject. Thus, in each of these methods, stent thrombosis may beintraprocedural stent thrombosis, acute stent thrombosis, sub-acutestent thrombosis, late stent thrombosis or very late stent thrombosis.Further, in each of these methods, the prevention of stent thrombosismay be prevention during percutaneous coronary intervention (PCI) orother vascular stent implantation.

In each of the relevant methods, mortality may be caused byintraprocedural stent thrombosis, acute stent thrombosis, sub-acutestent thrombosis, late stent thrombosis or very late stent thrombosis,or occlusion of a coronary artery.

Stent thrombosis may result from any means related to the implantation,presence, or maintenance of the stent in the vasculature of a subject.For example, stent thrombosis may be induced by implantation of a stent,such as a bare-metal stent, a drug-eluting stent, or other type of stentinto the subject. Similarly, stent thrombosis may develop over time dueto the presence of a stent, such as a bare-metal stent, a drug-elutingstent, or other type of stent in the subject. Thus, in each of theembodiments of the present invention stent thrombosis may beintraprocedural stent thrombosis, acute stent thrombosis (<24 hours postimplantation), sub-acute stent thrombosis (>24 hours and <30 days postimplantation), late stent thrombosis (>30 days and <12 months postimplantation) or very late stent thrombosis (>12 months postimplantation).

In each of the relevant methods, the prevention of stent thrombosis maybe prevention in the course of stent implantation during percutaneouscoronary intervention (PCI) or other vascular stent implantationprocedure.

In each of the relevant methods, the stent implantation may beimplantation of a bare-metal stent, a drug-eluting stent, or other typeof stent into a subject. The stent implantation is implantation duringpercutaneous coronary intervention (PCI) or other vascular stentimplantation. The mortality associated with stent implantation may bemortality due to intraprocedural stent thrombosis, acute stentthrombosis, sub-acute stent thrombosis, late stent thrombosis or verylate stent thrombosis.

The present invention includes a method of treating myocardialinfarction or reducing mortality in a subject experiencing myocardialinfarction. The course of treatment will generally follow diagnosis ofmyocardial infarction or at the onset of symptoms of myocardialinfarction. The pharmaceutical formulation may be a bolus intravenousdosage form or a continuous intravenous infusion dosage form, and may beadministered in combination with an oral dosage form. In preferredaspects, the pharmaceutical formulation is administered to the subjectwithin about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80 or 90 minutes of theonset of symptoms of myocardial infarction. The course of treatment maylast for a period of hours, days or weeks. The pharmaceuticalformulation may thus be administered to a subject to treat myocardialinfarction or to reduce mortality for about 1, 2, 3, 4, 5 or more hoursafter diagnosis of myocardial infarction or at the onset of symptoms ofmyocardial infarction, and be repeated for a number of days or weeks. Inparticular aspects, the pharmaceutical formulation may be administeredto the subject as an intravenous bolus, as a continuous intravenousinfusion, as an intravenous bolus followed by continuous intravenousinfusion, or some combination thereof, and optionally, in combinationwith an oral dosage form. In a particular example, the pharmaceuticalformulation is administered to the subject in a continuous intravenousinfusion dosage form over a period of at least about 0.5, 1, 1.5, 2,2.5, 3, 3.5 or 4 hours, or more. The treatment may be once, twice,thrice or more times a day, once every two days, once every three days,once every four days, once every five days, once every six days, once aweek, once every 10 days, once every two weeks, once every three weeks,once a month, or even less frequently.

In the methods of the invention directed to methods of reducingmortality in a subject experiencing myocardial infarction, mortality maybe reduced within a period of about 24, 36 or 48 hours after myocardialinfarction, within a period of about 30 days after myocardialinfarction, within a period of about six months after myocardialinfarction, or within a period of about one year after myocardialinfarction. In preferred embodiments, mortality is reduced by at leastabout 0.2%, 0.4%, 0.6%, 0.8%, 1.0% or 1.2% during the period incomparison to a subject not receiving cangrelor.

The present invention includes a method of preventing myocardialinfarction. The method comprises administration of a pharmaceuticalformulation of the present invention to a subject as a prophylaxisagainst myocardial infarction. Subjects appropriate for such preventionwould be any subject suspected of having a vascular thrombus, earlysymptoms of myocardial infarction or other disease or condition thatcould lead to myocardial infarction against which the pharmaceuticalformulations of the invention would be effective. The pharmaceuticalformulation may be in an oral dosage form, a bolus intravenous dosageform or a continuous intravenous infusion dosage form. In preferredaspects, the pharmaceutical formulation is administered to the subjectwithin about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80 or 90 minutes of whenearly or initial symptoms of myocardial infarction are detected. Thecourse of treatment may last for a period of hours, days or weeks. Thepharmaceutical formulation may thus be administered to a subject toprevent myocardial infarction for about 1, 2, 3, 4, 5 or more hoursafter early or initial symptoms of myocardial infarction are detected,and be repeated for a number of days or weeks. In particular aspects,the pharmaceutical formulation may be administered to the subjectorally, as an intravenous bolus, as a continuous intravenous infusion,as an intravenous bolus followed by continuous intravenous infusion, orsome combination thereof. In a particular example, the pharmaceuticalformulation is administered to the subject in a continuous intravenousinfusion dosage form over a period of at least about 0.5, 1, 1.5, 2,2.5, 3, 3.5 or 4 hours, or more. The treatment may be once, twice,thrice or more times a day, once every two days, once every three days,once every four days, once every five days, once every six days, once aweek, once every 10 days, once every two weeks, once every three weeks,once a month, or even less frequently.

In each of the relevant methods, myocardial infarction may be any formof myocardial infarction, including acute myocardial infarction (firstfew hours to 7 days), healing myocardial infarction (7 to 28 days),healed myocardial infarction (29 days and beyond), acute non-ST-elevatedmyocardial infarction and acute ST-elevated myocardial infarction.Myocardial infarction may be induced by any mechanism, includingimplantation of a bare-metal stent or a drug-eluting stent into thesubject, or other vascular stent implantation, or arise duringpercutaneous coronary intervention (PCI). Myocardial infarction may alsobe caused by intraprocedural stent thrombosis, acute stent thrombosis,sub-acute stent thrombosis, late stent thrombosis, very late stentthrombosis or occlusion of a coronary artery. Mortality may be caused byintraprocedural stent thrombosis, acute stent thrombosis, sub-acutestent thrombosis, late stent thrombosis or very late stent thrombosis,or occlusion of a coronary artery.

Subjects

As used herein, a “subject” upon which the methods of the presentinvention may be practiced refers to an animal, such as a mammalian oran avian species, including a human, a non-human primate, a horse, acow, a sheep, a goat, a dog, and a cat.

To further characterize the subjects to which the methods of the presentinvention may be applied, it is noted that the subject may have suffereda stroke, or the subject may not have suffered a stroke. The subject mayhave diabetes mellitus, or the subject may not have diabetes mellitus.The subject may have hypertension, or the subject may not havehypertension. The subject may have hyperlipidemia, or the subject maynot have hyperlipidemia. The subject may have suffered a myocardialinfarction, or the subject may not have suffered a myocardialinfarction. The subject may have a family history of coronary arterydisease (CAD), or the subject may not have a family history of CAD. Thesubject may have undergone percutaneous transluminal coronaryangioplasty (PTCA), or the subject may not have undergone PTCA. Thesubject may have undergone percutaneous coronary intervention (PCI), orthe subject may not have undergone PCI. The subject may have undergonecoronary artery bypass graft (CABG), or the subject may not haveundergone CABG. The subject may have congestive heart failure, or thesubject may not have congestive heart failure. The subject may haveperipheral arterial disease (PAD), or the subject may not have PAD.

In certain aspects, the subject may have stent thrombosis, be at risk ofdeveloping stent thrombosis, or be undergoing stent implantation. Thesubject may have stent thrombosis in more than one artery or vein. Thus,the subjects encompassed by the methods of the present invention includesubjects undergoing vascular stent implantation and subjects havingundergone vascular stent implantation.

In certain aspects, the subject may be undergoing coronary artery bypassgrafting (CABG) surgery or about to undergo CABG surgery (e.g., in lessthan 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day). Such subjects may have anacute coronary syndrome (AC S) and/or have been treated with a coronarystent. Such patients may also have been receiving thienopyridinetreatment prior to treatment using one of the methods of the presentinvention. For example, the subject may be treated using a method of thepresent invention as a “bridge” between cessation of oral antiplatelettherapy and the beginning of cardiac surgery.

Results of the Methods

Each of the methods recited in the present invention may include theadditional step of measuring the effect or effectiveness of thepharmaceutical formulation during or after administration. In oneexample, the additional step of measuring an effect of thepharmaceutical formulation may be performed during or about 0.5, 1, 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20 or 24 hours, or more, aftercompletion of a method of the invention. The effects that may bemeasured in the methods of the present invention include a change in thelevel of platelet reactivity, an increase in luminal diameter within thestent, a decrease in the size of the stent thrombus, and a decreasedincidence of myocardial infarction. Each of these effects woulddemonstrate the effectiveness of the compounds comprising thepharmaceutical composition.

The invention will now be further described by way of the followingnon-limiting examples, which further illustrate the invention, and arenot intended, nor should they be interpreted to, limit the scope of theinvention.

EXAMPLES Example 1 Solution Stability of Cangrelor Tetrasodium

Given the nature of cangrelor as an anhydride, its stability in aqueoussolution was determined.

Solutions of cangrelor in water at 1 mg/mL were stored at 4° C., 25° C.and 40° C. for 8 days and protected from light. The levels of theimpurities were determined by reverse phase HPLC and reported in Table1.

TABLE 1 Solution stability of Cangrelor (1 mg/mL) in purified water for8 days Measured level (% w/w) After 8 days under storage conditionsdescribed 4° C. and 25° C. and 60% 40° C. and ambient relative ambientMeasured analyte Initial humidity humidity humidity Cangrelor 98.2398.45 96.94 90.46 Impurity A 0.16 0.22 1.58 7.50 Impurity D ND ND ND NDImpurity C 0.28 0.26 0.27 0.37 Total Impurities* 1.76 1.54 3.06 9.56ND—none detected. *includes all impurities including those formed duringthe synthesis of cangrelor.

The results demonstrate that cangrelor is a moisture-sensitive molecule,in particular through hydrolysis to impurity A. Therefore, it isimportant to control the moisture content of the solid cangrelor.

Example 2 Accelerated pH Stability of High Purity Cangrelor

The stability of cangrelor drug substance in aqueous solution wasinvestigated over a range of pH values. The extent of degradation wasdetermined using a reverse phase HPLC method. The effect of pH on thestability of cangrelor in aqueous solution at 1 mg/mL was studied over apH range of 1 to 12 and the solutions were stored at 40° C. for 7 daysprotected from light (Table 2).

TABLE 2 Stability of Cangrelor in various pH buffer solutions for 7 daysat 40° C. protected from light Impurity A Impurity D Total Impurities*pH Medium (% w/w) (% w/w) (% w/w) 1.0 0.1M HCl 28.51 68.12 97.28 3.0Phosphate 28.69 0.81 31.48 5.0 Phosphate 20.70 ND 22.16 7.0 Phosphate14.31 ND 15.71 9.0 Phosphate 3.25 ND 4.90 11.0 Phosphate 2.61 ND 4.2812.0 0.1M NaOH 2.42 ND 4.39 ND—none detected. *Sum of all impuritiesincluding degradants and impurities occurring during the synthesis ofcangrelor.

The degradation of cangrelor after 7 days at 40° C. was pH dependent andoccurred primarily via hydrolysis of cangrelor to form either impurity A(hydrolysis of the dichloromethylenebisphosphonic acid group oncangrelor) or impurity D (hydrolysis of the glycosidic bond oncangrelor) or both impurity A and impurity D. Based on the pH stabilitydata, it is evident that cangrelor is more sensitive to hydrolysis inacidic pH but progressively more stable in the alkaline pH range. Themain route of degradation at lower pH (1-3) is the hydrolysis of theglycosidic bond to form impurity D, which is expected on the basis ofthe lability of glycosidic bonds to acid hydrolysis. This particulardegradation pathway was not detected at pH 5 or higher. On the otherhand, there is a reduction in the rate of the hydrolysis pathway leadingto impurity A with increasing pH.

While these conditions (40° C. for 7 days) are not representative ofstorage conditions for the pharmaceutical formulation comprisingcangrelor, they provide for a convenient way to evaluate the impact ofpH on the degradation of cangrelor. While the results clearlydemonstrate a higher pH will provide favorable stability, it isdesirable to have a final drug product pH at or close to physiologicalpH. Therefore, it is important to design a formulation that providesacceptable stability at or close to physiological pH.

Example 3 Photostability of Cangrelor in Solution

Impurity C is obtained through the formation of sulfoxide from a sulfidefound in cangrelor and is therefore an oxidized form of cangrelor. Suchoxidations are mediated by light (Liang et al. J. Am. Chem. Soc. 1983,105, 4717).

The photostability of cangrelor was measured to evaluate the potentialfor light mediated degradation. Namely, solid cangrelor was placed intwo quartz cuvettes. Both cuvettes were placed in a chamber and exposedto a combination of 320-400 nM (near UV) and 400-800 nM (visible) lightfor a total of 7.8×10⁶ LUX hours and 221 watt hours/m², but one cuvettewas wrapped in aluminum foil. After 17 days, the levels of impurities inthe sample were determined by reverse phase HPLC. In this study, thetotal level of impurities, initially at 0.8% (w/w) in this batch, wasfound to be at 4.3% (w/w) in the exposed sample and 1.2% (w/w) in theshielded sample. While the degradation of the unexposed sample accountsfor degradation that is not light mediated, the higher rate ofdegradation in the exposed sample demonstrates the sensitivity ofcangrelor to light mediated processes.

While this study is not representative of the conditions of storage ofcangrelor in a quantitative way, it does show qualitatively thatcangrelor is sensitive to conditions of exposure to air and light.

While the previous study demonstrated the need to protect cangreloritself from light and air, a separate study was performed with the bulkformulation of cangrelor prepared as per the process described in theinvention. The bulk formulation was dissolved at a concentration of16.42 mg/mL and exposed to ordinary room lighting for 4, 8, and 24 hoursand then analyzed for assay and impurities. Solution not exposed tolight served as the control. The impurity levels were determined byreverse phase HPLC and presented in Table 3.

TABLE 3 Light stability data of Cangrelor bulk formulation CangrelorImpurity level level (% initial) (% peak area) Time Point Shielded fromExposed to Shielded from Exposed to (hour) light light Impurities lightlight Initial 100% N/A Impurity A 0.13 N/A Impurity B <0.05 N/A 4 98.9101.5 Impurity A 0.13 0.13 Impurity B 0.06 0.06 Impurity C NP <0.05 8100.7 100 Impurity A 0.14 0.14 Impurity B 0.06 0.06 Impurity C NP 0.0724 97.8 99.1 Impurity A 0.16 0.16 Impurity B 0.06 NP Impurity C NP 0.14N/A—not applicable NP—not present

In this study, even over the short duration of the experiment, it isclear that the level of impurity C increases over time. This clearlydemonstrates that cangrelor is sensitive to photooxidation.

Example 4 Sensitivity of Cangrelor to Oxidation

The susceptibility of cangrelor to oxidation was evaluated by exposingcangrelor formulated in mannitol/sorbitol to 0.1% hydrogen peroxide for1 h. The levels of cangrelor and impurity C (the oxidation product ofcangrelor) were then measured by reverse-phase HPLC. Even under theserelatively mild oxidative conditions, there was only 12.46±0.70% ofcangrelor by peak area left after 1 h and 83.88±0.47% of impurity C hadbeen produced by peak area.

This experiment shows that cangrelor is susceptible to oxidation andwhile these conditions are harsher than those brought by exposure toair, they demonstrate the need to keep cangrelor away from oxidants suchas oxygen.

Example 5 Correlation of Stability with Moisture Level

The hygroscopicity of cangrelor tetrasodium has been measured using thedynamic vapor adsorption analysis method and cangrelor was determined tobe hygroscopic. Given the fact that as seen in Examples 1 and 2,cangrelor is sensitive to hydrolysis, it was of interest to determine ifabsorbed moisture can cause degradation over time.

Vials of cangrelor were prepared through the lyophilization of cangrelortetrasodium (57.72 mg per vial), mannitol (164.4 mg per vial) andsorbitol (54.3 mg per vial) in two batches. Batch A was lyophilized to amoisture content of 0.33% and batch B was lyophilized to a moisturecontent of 2.0%. The vials were sealed with a rubber cap, crimped andstored in one of four conditions: at 5° C. and ambient humidity, at 25°C. and 60% relative humidity, at 30° C. and 60% relative humidity and at40° C. and 75% relative humidity. At specific time points ranging from0-12 months, the levels of impurities were measured by reverse phaseHPLC and the moisture levels were measured by Karl-Fischer titration.The values measured are reported in Table 4.

TABLE 4 Long term stability of cangrelor formulations Storage conditionsTotal (° C./% Relative Moisture level impurities Batch Humidity) Time(months) (% w/w) (% w/w) A Initial 0 0.33 0.18 5/ambient 3 0.36 0.19 60.53 0.18 9 0.58 0.19 12 0.74 0.18 25/60 3 0.57 0.2 6 0.72 0.19 9 0.770.19 12 0.84 0.19 30/60 3 0.62 0.2 6 0.68 0.21 9 0.75 0.22 12 0.82 0.2340/75 3 0.66 0.27 6 0.78 0.37 9 1.08 0.45 12 1.01 0.70 B Initial 0 2.000.18 5/ambient 3 1.80 0.19 6 2.03 0.18 9 2.21 0.18 12 2.18 0.18 25/60 32.06 0.19 6 1.93 0.22 9 2.11 0.24 12 2.08 0.25 30/60 3 1.94 0.24 6 1.860.28 9 2.30 0.31 12 1.90 0.31 40/75 3 1.81 0.52 6 1.78 3.64 9 2.34 5.8012 2.05 5.75

This data, and in particular the values measured at 40° C. and 75%relative humidity, clearly demonstrates that even in sealedlyophilization vials, the cangrelor formulation slowly absorbs moistureand that, in parallel, the quantity of impurities rise. It shows that aprocess to exclude moisture is necessary to produce a cangrelorcomposition that can be stored for a period and remain useable.

Example 6 Stability of Cangrelor Produced and Stored Under Conditions ofthe Invention

Cangrelor batches A-E (Table 5) were produced according to theinvention. Their pH was adjusted to 8.5 prior to lyophilization and theywere stored in glass vials stoppered with a stopper specificallyselected for its ability to retain as little moisture as possible afterautoclaving and after drying for 8 h at 105° C. Together with cangrelor(50 mg of the tetrasodium salt per vial), excipients mannitol (164.4 mgper vial) and sorbitol (54.3 mg per vial) were included in theformulation.

Cangrelor lots API A-B are cangrelor tetrasodium. They were stored indouble polyethylene bags in HDPE pails.

These lots were placed in storage at 25° C. and 60% relative humidityand at the time points indicated in Table 5, aliquots were removed andthe level of impurities was measured by either reverse phase HPLC or byion chromatography for impurity E. In addition their moisture contentwas determined by Karl-Fischer titration. All these data are reported inTable 5. In addition, throughout the storage period, the pH of 1% w/vsolutions of the material in batches A-E were measured and remained atbetween 8.4 and 8.8 throughout.

TABLE 5 Long term stability of cangrelor formulations Analyteconcentration (% w/w) over time (months) Lot Analyte Initial 1 3 6 9 1218 24 30 36 Batch A Impurity A 0.25 0.25 0.26 0.26 0.26 0.27 0.28 0.290.30 0.31 Impurity B 0.06 0.06 0.06 0.06 0.06 0.06 0.07 0.06 0.06 0.07Impurity C <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 0.05 <0.05 <0.05 0.05Impurity E 0.09 0.10 0.10 0.10 0.08 0.08 0.09 0.08 0.09 0.09 Total 0.700.70 0.70 0.80 0.66 0.67 0.70 0.70 0.80 0.80 impurities Moisture 0.3 0.40.4 0.4 0.4 0.6 0.6 0.6 0.8 0.8 Batch B Impurity A 0.13 0.13 0.12 0.140.14 0.14 0.19 0.16 0.19 — Impurity B <0.05 <0.05 <0.05 <0.05 <0.05<0.05 <0.05 <0.05 0.0 — Impurity C <0.05 <0.05 <0.05 <0.05 <0.05 <0.05<0.05 0.05 0.06 — Impurity E <0.05 <0.05 <0.05 <0.05 <0.05 0.05 <0.05<0.05 0.04 — Total 0.13 0.13 0.13 0.14 0.14 0.19 0.19 0.36 0.40 —impurities Moisture 0.3 0.3 0.4 0.4 0.5 0.5 0.5 0.6 0.7 — Batch CImpurity A 0.10 0.15 0.15 0.15 0.16 0.16 0.17 — — — Impurity B <0.05<0.05 <0.05 0.05 <0.05 <0.05 0.05 — — — Impurity C <0.05 <0.05 <0.050.05 0.04 0.05 0.06 — — — Impurity E <0.05 <0.05 <0.05 0.05 <0.05 <0.05<0.05 — — — Total 0.10 0.15 0.15 0.30 0.21 0.21 0.30 — — — impuritiesMoisture 0.3 0.4 0.4 0.5 0.5 0.7 1.1 — — — Batch D Impurity A ND 0.100.12 0.13 0.14 0.13 0.13 0.16 — — Impurity B <0.05 — — 0.05 0.05 0.05 —0.05 — — Impurity C <0.05 <0.05 <0.05 <0.05 ND ND <0.05 0.05 — —Impurity E <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 — — Total0.50 0.20 0.20 0.20 0.20 0.20 0.30 0.30 — — impurities Moisture 0.3 0.40.3 0.5 0.4 0.6 0.6 0.7 — — Batch E Impurity A ND 0.10 0.11 0.12 0.140.11 0.12 — — — Impurity B — — 0.05 0.05 0.05 0.05 0.05 — — — Impurity C— — <0.05 ND ND 0.05 0.05 — — Impurity E <0.05 <0.05 <0.05 <0.05 <0.05<0.05 <0.05 — — Total 0.10 — 0.20 0.20 0.20 0.20 0.40 — — impuritiesMoisture 0.4 — 0.4 0.4 0.7 0.7 0.7 API A Impurity A 0.15 0.28 0.47 — — —— — — — Impurity B 0.11 0.22 0.33 — — — — — — — Impurity C — — — — — — —— — — Impurity E 0.06 0.08 0.11 — — — — — — — Total 0.70 1.10 1.40 — — —— — — — impurities Moisture 4.3 7.7 11.3 — — — — — — — API B Impurity A0.31 0.36 0.51 0.70 — — — — — — Impurity B 0.11 0.19 0.32 0.48 — — — — —— Impurity C <0.05 0.06 0.08 0.19 — — — — — — Impurity E — — — — — — — —— — Total 0.80 1.10 1.50 1.90 — — — — — — impurities Moisture 7.0 6.99.9 11.6 — — — — — —

These data demonstrate that batches A-E produced by the processdisclosed in the invention remain stable for up to 36 months withouteither significant degradation or significant increase in moisturecontent. In comparison, lots API A and API B rapidly concentratemoisture and display significant degradation over time.

These data support the use of the process described in the invention forthe generation of high purity cangrelor formulations that can be storedfor a long period of time and be useable in patients.

What is claimed is:
 1. A pharmaceutical formulation comprising highpurity cangrelor, or a salt thereof, as an active ingredient and one ormore pharmaceutically acceptable excipients, wherein the pharmaceuticalformulation has a pH of between about 7.0 and about 9.5, wherein thehigh purity cangrelor or salt thereof has a combined total of selectedhydrolysis and oxidation degradants of cangrelor not exceeding about1.5% by weight of the high purity cangrelor, and wherein the selectedhydrolysis and oxidation degradants are one or more members selectedfrom the group consisting of:


2. The pharmaceutical formulation of claim 1, wherein the combined totalof selected hydrolysis and oxidation degradants of cangrelor does notexceed about 1.3% by weight of the high purity cangrelor.
 3. Thepharmaceutical formulation of claim 1, wherein the amount of impurity Ais less than about 0.5% by weight, the amount of impurity B present isless than about 0.2% by weight, the amount of impurity C is less thanabout 0.3% by weight, the amount of impurity D is less than about 0.2%by weight, and the amount of impurity E is less than about 0.5% byweight of the high purity cangrelor.
 4. The pharmaceutical formulationof claim 1, wherein the maximum impurity level of impurities A and D iseach less than about 0.5% by weight of the high purity cangrelor.
 5. Thepharmaceutical formulation of claim 1, wherein the pharmaceuticallyacceptable excipient is a polyol.
 6. The pharmaceutical formulation ofclaim 1, wherein the pharmaceutically acceptable excipients are mannitoland sorbitol.
 7. The pharmaceutical formulation of claim 1, wherein thepharmaceutical formulation comprises about 16-21% high purity cangrelorand about 84-79% of the one or more pharmaceutically acceptableexcipients, by weight of the pharmaceutical formulation.
 8. A sealedvessel containing the pharmaceutical formulation of claim 1 under achemically inert dry gas.
 9. The sealed vessel of claim 8, wherein thechemically inert dry gas is nitrogen or argon.
 10. The sealed vessel ofclaim 8, wherein moisture in the pharmaceutical formulation remainsbelow 5.0% on a weight basis over a period of at least about 6 months.11. The sealed vessel of claim 8, wherein after a period of about 12months, the pharmaceutical formulation is characterized by a pH ofbetween about 7.0 and 9.5 for a 1% solution by weight, an amount ofmoisture less than about 5% on a weight basis, a maximum level of theimpurities A, B, C and D not exceeding about 1% each by weight of thehigh purity cangrelor and a maximum level of impurity E not exceedingabout 0.5% by weight of the high purity cangrelor.
 12. The sealed vesselof claim 8, wherein after a period of about 12 months, thepharmaceutical formulation is characterized by a pH of between about 7.0and 9.5 for a 1% solution by weight, an amount of moisture less thanabout 5% on a weight basis, and a maximum combined level of impuritiesA, B, C, D and E not exceeding about 5% by weight of the high puritycangrelor.
 13. The sealed vessel of claim 8, wherein after a period ofabout 12 months, the pharmaceutical formulation is characterized by a pHof between about 7.0 and 9.5 for a 1% solution by weight, an amount ofmoisture less than about 5% on a weight basis, and a maximum combinedlevel of impurities A, B, C, D and E not exceeding about 2% by weight ofthe high purity cangrelor.
 14. A pharmaceutical formulation consistingof high purity cangrelor, or a salt thereof, as an active ingredient andmannitol and/or sorbitol as a pharmaceutically acceptable excipient,wherein the pharmaceutical formulation has a pH of between about 7.0 andabout 9.5, wherein the high purity cangrelor or salt thereof has acombined total of selected hydrolysis and oxidation degradants ofcangrelor not exceeding about 1.5% by weight of the high puritycangrelor, and wherein the selected hydrolysis and oxidation degradantsare one or more members selected from the group consisting:


15. The pharmaceutical formulation of claim 14, wherein the combinedtotal of selected hydrolysis and oxidation degradants of cangrelor doesnot exceed about 1.3% by weight of the high purity cangrelor.
 16. Thepharmaceutical formulation of claim 14, wherein the amount of impurity Ais less than about 0.5% by weight, the amount of impurity B present isless than about 0.2% by weight, the amount of impurity C is less thanabout 0.3% by weight, the amount of impurity D is less than about 0.2%by weight, and the amount of impurity E is less than about 0.5% byweight of the high purity cangrelor.
 17. The pharmaceutical formulationof claim 14, wherein the maximum impurity level of impurities A and D iseach less than about 0.5% by weight of the high purity cangrelor. 18.The pharmaceutical formulation of claim 14, wherein the pharmaceuticallyacceptable excipient is a polyol.
 19. The pharmaceutical formulation ofclaim 14, wherein the pharmaceutically acceptable excipients aremannitol and sorbitol.
 20. The pharmaceutical formulation of claim 14,wherein the pharmaceutical formulation comprises about 16-21% highpurity cangrelor and about 84-79% of the one or more pharmaceuticallyacceptable excipients, by weight of the pharmaceutical formulation. 21.A sealed vessel containing the pharmaceutical formulation of claim 14under a chemically inert dry gas.
 22. The sealed vessel of claim 21,wherein the chemically inert dry gas is nitrogen or argon.
 23. Thesealed vessel of claim 21, wherein moisture in the pharmaceuticalformulation remains below 5.0% on a weight basis over a period of atleast about 6 months.
 24. The sealed vessel of claim 21, wherein after aperiod of about 12 months, the pharmaceutical formulation ischaracterized by a pH of between about 7.0 and 9.5 for a 1% solution byweight, an amount of moisture less than about 5% on a weight basis, amaximum level of the impurities A, B, C and D not exceeding about 1%each by weight of the high purity cangrelor and a maximum level ofimpurity E not exceeding about 0.5% by weight of the high puritycangrelor.
 25. The sealed vessel of claim 21, wherein after a period ofabout 12 months, the pharmaceutical formulation is characterized by a pHof between about 7.0 and 9.5 for a 1% solution by weight, an amount ofmoisture less than about 5% on a weight basis, and a maximum combinedlevel of impurities A, B, C, D and E not exceeding about 5% by weight ofthe high purity cangrelor.
 26. The sealed vessel of claim 21, whereinafter a period of about 12 months, the pharmaceutical formulation ischaracterized by a pH of between about 7.0 and 9.5 for a 1% solution byweight, an amount of moisture less than about 5% on a weight basis, anda maximum combined level of impurities A, B, C, D and E not exceedingabout 2% by weight of the high purity cangrelor.